Methods and Compositions Using Klotho-FGF Fusion Polypeptides

ABSTRACT

The present invention is directed to methods, kits and compositions for preventing or treating age-related conditions or metabolic disorders. The Klotho fusion polypeptides of the invention include at least a Klotho protein or an active fragment thereof. The Klotho fusion proteins are useful in the treatment and prevention of a variety of age-related conditions and metabolic disorders.

This application claims priority to U.S. Provisional Application Ser.No. 61/063,015 filed 28 Jan. 2008, the contents of which areincorporated herein by reference in their entirety.

1. BACKGROUND

The alpha-Klotho gene encodes a 130 kDa single pass type I transmembraneprotein with an extracellular domain and a short cytoplasmic domain. Theextracellular domain of alpha-Klotho protein comprises two subdomainstermed, KL-D1 and KL-D2. These two subdomains share sequence homology toβ-glucosidase of bacteria and plants. The extracellular domain of thealpha-Klotho protein may be bound to the cell surface by thetransmembrane domain or may be cleaved and released into theextracellular milieu. Cleavage of the extracellular domain appears to befacilitated by local low extracellular Ca²⁺ concentrations.

In addition to alpha-Klotho, a homolog of alpha-Klotho, beta-Klotho, hasbeen identified (Ito et al., Mech. Dev. 98:115-9 (2000)). Beta-Klotho isalso a single pass type I transmembrane protein with extracellular KL-D1and KL-D2 subdomains.

Modulation of alpha-Klotho expression has been demonstrated to produceaging related characteristics in mammals. Mice homozygous for a loss offunction mutation in the alpha-Klotho gene develop characteristicsresembling human aging, including shortened lifespan, skin atrophy,muscle wasting, arteriosclerosis, pulmonary emphysema and osteoporosis(Kuro-o et al., Nature, 390:45-51 (1997)). In contrast, overexpressionof the alpha-Klotho gene in mice extends lifespan and increasesresistance to oxidative stress relative to wild-type mice (Kurosu etal., Science 309:1829-1833 (2005); Yamamoto et al., J. Biol. Chem.280:38029 38034 (2005)).

Fibroblast growth factors (FGFs) constitute a family of homologouspolypeptide growth factors expressed in many organisms (Ornitz and Itoh,Genome Biol. 2: reviews, 3005.1-3005.12 (2001)). Among vertebratespecies, FGFs are highly conserved in both gene structure and amino-acidsequence, having between 13-71% amino acid identity with one another. Inhumans, there are 22 known members of the FGF family (FGF15 is the mouseortholog of human FGF19, hence there is no human FGF15). During earlydevelopment, FGFs regulate cell proliferation, migration, anddifferentiation, but in the adult organism, FGFs maintain homeostasis,function in tissue repair, and respond to injury.

FGFs function as growth factors by binding and thereby activatingcell-surface FGF receptors. FGF receptors (FGFRs) are tyrosine kinasereceptors that activate signal transduction through autophosphorylationof FGFR, phosphorylation of FRS2 (FGF receptor substrate 2) and ERK1/2(extracellular signal-regulated protein kinase ½), and activating Egr-1(early growth response-1). FGFs also have a high affinity for heparinsulfate proteoglycans. When bound to FGFs, heparin sulfate enhances theactivation of FGFRs.

Recent studies have demonstrated strikingly similar biologicalcharacteristics between FGF23-deficient mice and alpha-Klotho-deficientmice (Shimada et al., J. Clin. Invest. 113:561-568 (2004); Yoshida etal. Endocrinology 143:683-689 (2002)), indicating functional crosstalkbetween FGF23 and alpha-Klotho. These studies led to the identificationof alpha-Klotho as an obligatory partner of FGF23, in terms of bothbinding and signaling through its cognate FGF receptors (Urakawa et al.,Nature 22:1524-6 (2007)). The alpha-Klotho gene is mainly expressed inkidney, parathyroid gland and choroid plexus. It is hypothesized thatthe tissue-specific expression of alpha-Klotho restricts activation ofFGF23 signaling to those tissues.

Similar to FGF23/alpha-Klotho, beta-Klotho is an obligatory partner ofFGF19 and FGF21, both in binding and in signaling through theirrespective cognate FGF receptors (Ogawa et al., Proc. Natl. Acad. Sci.USA 104:7432-7 (2007); Lin et al., J. Biol. Chem. 282:27227-84 (2007);and Wu et al., J. Biol. Chem. 282:29069-72 (2007)). Such studies havealso demonstrated the involvement of beta-Klotho in regulatingtissue-specific metabolic activity. Beta-Klotho was initially shown toact with FGF21 as a cofactor for regulating carbohydrate and lipidmetabolism in adipose tissue. Beta-Klotho in conjunction with FGF19regulates bile acid metabolism in liver, thus explaining elevated bilesynthesis in beta-Klotho deficient mice (Ito et al., J Clin Invest. 2005August; 115(8):2202-8).

U.S. Pat. No. 6,579,850 describes polypeptides and compositionscomprising an alpha-Klotho polypeptide. Human and mouse alpha-Klothopolypeptides are disclosed. The patent also disclosed that compositionscomprising the polypeptides are useful in treating a syndrome resemblingpremature aging, treating adult diseases, and suppressing aging.

U.S. Pat. No. 7,223,563 describes isolated nucleic acids encoding theFGF23 polypeptide sequence or recombinant cells comprising such anisolated nucleic acid. The patent further relates to methods ofdiagnosing and treating hypophosphatemic and hyperphosphatemicdisorders, osteoporosis, dermatomyositis, and coronary artery disease.

U.S. Pat. No. 7,259,248 describes isolated nucleic acids encoding theFGF21 polypeptide sequence. The patent further relates to methods ofdiagnosing and treating liver disease, conditions related to thymicfunction, and methods of treating conditions of the testis.

2. SUMMARY OF THE INVENTION

The present invention is directed to methods, kits and compositions forpreventing or treating age-related conditions or metabolic disorderswith Klotho fusion polypeptides or soluble Klotho polypeptides. TheKlotho fusion polypeptides of the present invention are formed of aKlotho protein or an active fragment thereof (e.g., sKlotho). In someembodiments, the present invention provides a Klotho fusion polypeptidecomprising a Klotho protein or an active fragment thereof and afibroblast growth factor or an active fragment thereof.

In a first aspect, the invention provides a fusion polypeptide having atleast one extracellular subdomain of a Klotho protein and a fibroblastgrowth factor or an active fragment thereof. The Klotho extracellulardomain may be derived from either the alpha or beta Klotho isoforms.Further, although the FGF component of the Klotho fusion polypeptide isdescribed primarily with reference to fibroblast growth factor-19,fibroblast growth factor-21 and fibroblast growth factor-23, it iscontemplated that any of the twenty-three known FGFs can be used inpracticing the invention. The reader of the instant application mayassume that each of every combination of alpha or beta extracellulardomain with each human FGF protein or an active fragment thereof areindividually and specifically contemplated.

According to the present invention, the extracellular domain of theKlotho protein can include one or both of the KL-D1 and KL-D2 domains ofa Klotho protein. In some embodiments, the Klotho fusion polypeptide ofthe invention has at least two extracellular subdomains of a Klothoprotein. For example, the two extracellular subdomains can be two KL-D1domains in tandem repeats, two KL-D2 domains in tandem repeats, or oneKL-D1 domain and one KL-D2 domain. In one embodiment, the fusionpolypeptide of the invention comprises amino acids 28-292 of the fulllength alpha Klotho protein. In another embodiment, the fusionpolypeptide of the invention comprises amino acids 52-997 of the fulllength beta Klotho protein.

According to the present invention, a polypeptide comprising at leastone extracellular subdomain of a Klotho protein and a FGF or an activefragment thereof may be linked together covalently, for example,chemically linked or fused in frame by a peptide bond. They may alsolinked via a linker. Non-limiting examples of polypeptide linker are SEQID NOs:11, 12, 13, 14, 15, 16, 17, and 18. Such linkers may comprise atleast one and up to about 30 repeats of SEQ ID NOs:11, 12, 13, 14, 15,16, 17 and 18.

According to the present invention, the extracellular subdomain of aKlotho protein and the fibroblast growth factor can be operativelylinked to one another in a variety of orientations and manners. Forexample, the extracellular subdomain of the Klotho protein can beoperatively linked to the N-terminus of the fibroblast growth factor oralternatively the fibroblast growth factor can be operatively linked tothe N-terminus of an extracellular subdomain of the Klotho protein.

In one embodiment, the present invention provides a fusion polypeptidecomprising a sKlotho of a Klotho protein and a linker. In anotherembodiment, the present invention provides a fusion polypeptidecomprising a sKlotho of the alpha Klotho protein and a linker. Inanother embodiment, the present invention provides a fusion polypeptidecomprising a sKlotho of the beta Klotho protein and a linker. In yetanother embodiment, the present invention provides a human FGF proteinor an active fragment thereof (e.g., without signal peptide) and alinker. Pharmaceutical compositions comprising the fusion proteins ofthe invention and their uses for treating or preventing age-relatedconditions or metabolic disorders are also encompassed by the presentinvention.

In one embodiment, the present invention provides a fusion polypeptidecomprising a sKlotho of alpha Klotho protein with signal peptide fused(directly or indirectly via a linker) to FGF-23. In another embodiment,the present invention provides a fusion polypeptide comprising a sKlothoof alpha Klotho protein without signal peptide fused (directly orindirectly via a linker) to FGF-23. In another embodiment, the presentinvention provides sKlotho of alpha Klotho protein with signal peptidefused (directly or indirectly via a linker) to FGF-23 without signalpeptide. In another embodiment, the present invention provides a fusionpolypeptide comprising sKlotho of alpha Klotho protein without signalpeptide fused (directly or indirectly via a linker) to FGF-23 withoutsignal peptide.

In one embodiment, the present invention provides a fusion polypeptidecomprising a sKlotho of alpha Klotho protein with signal peptide fused(directly or indirectly via a linker) to FGF-23 (R179Q) variant. Inanother embodiment, the present invention provides a fusion polypeptidecomprising a sKlotho of alpha Klotho protein without signal peptidefused (directly or indirectly via a linker) to FGF-23 (R179Q) variant.In another embodiment, the present invention provides sKlotho of alphaKlotho protein with signal peptide fused (directly or indirectly via alinker) to FGF-23 (R179Q) variant without signal peptide. In anotherembodiment, the present invention provides a fusion polypeptidecomprising sKlotho of alpha Klotho protein without signal peptide fused(directly or indirectly via a linker) to FGF-23 (R179Q) variant withoutsignal peptide.

In one embodiment, the present invention provides a fusion polypeptidecomprising (1) sKlotho of alpha Klotho protein with signal peptide; (2)a linker; and (3) FGF-23 (R179Q) variant without signal peptide. Inanother embodiment, the present invention provides a fusion polypeptidecomprising (1) sKlotho of alpha Klotho protein without signal peptide;(2) a linker; and (3) FGF-23 (R179Q) variant without signal peptide. Insome embodiments, the fusion polypeptides of the invention areglycosylated.

In one embodiment, the present invention provides a fusion polypeptidecomprising (1) sKlotho of alpha Klotho protein with signal peptide (SEQID NO: 44 or SEQ ID NO:45); (2) a linker comprising SEQ ID NO:11; and(3) FGF-23 (R179Q) variant without signal peptide (SEQ ID NO: 43). Inanother embodiment, the present invention provides a fusion polypeptidecomprising (1) sKlotho of alpha Klotho protein without signal peptide(SEQ ID NO:7); (2) a linker comprising SEQ ID NO:11; and (3) FGF-23(R179Q) variant without signal peptide (SEQ ID NO: 43). In oneembodiment, the present invention provides a fusion polypeptidecomprising the amino acid sequence of SEQ ID NO:19, 20, 40, or 41. Insome embodiments, the fusion polypeptides of the invention areglycosylated.

In one embodiment, the present invention provides a fusion polypeptidecomprising sKlotho of alpha Klotho protein with signal peptide (SEQ IDNO:44 or SEQ ID NO:45); and a linker comprising SEQ ID NO:11. In anotherembodiment, the present invention provides a fusion polypeptidecomprising sKlotho of alpha Klotho protein without signal peptide (SEQID NO:7); and a linker comprising SEQ ID NO:11. In some embodiments, thefusion polypeptides of the invention are glycosylated.

In one embodiment, the present invention provides a fusion polypeptidecomprising a human FGF protein or an active fragment thereof (e.g.,without the signal peptide); and a linker comprising SEQ ID NO:11. Insome embodiments, the fusion polypeptides of the invention areglycosylated.

In one embodiment, the present invention provides a pharmaceuticalcomposition (e.g., in an intra-muscular administering form) comprising(e.g., as a sole pharmaceutically active ingredient) a fusionpolypeptide (e.g., glycosylated or non-glycosylated) that comprises (1)sKlotho of alpha Klotho protein with signal peptide (SEQ ID NO: 44 orSEQ ID NO:45); (2) a linker comprising SEQ ID NO:11; and (3) FGF-23(R179Q) variant without signal peptide (SEQ ID NO: 43); and uses of thepharmaceutical composition for treating and/or preventing age-relatedconditions, such as muscular atrophy. In another embodiment, the presentinvention provides a pharmaceutical composition (e.g., in anintra-muscular administering form) comprising (e.g., as a solepharmaceutically active ingredient) a fusion polypeptide (e.g.,glycosylated or non-glycosylated) that comprises (1) sKlotho of alphaKlotho protein without signal peptide (SEQ ID NO:7); (2) a linkercomprising SEQ ID NO:11; and (3) FGF-23 (R179Q) variant without signalpeptide (SEQ ID NO: 43); and uses of the pharmaceutical composition fortreating and/or preventing age-related conditions, such as muscularatrophy. In one embodiment, the present invention provides apharmaceutical composition (e.g., in an intra-muscular administeringform) comprising (e.g., as a sole pharmaceutically active ingredient) afusion polypeptide (e.g., glycosylated or non-glycosylated) comprisingthe amino acid sequence of SEQ ID NO:19, 20, 40, or 41; and uses of thepharmaceutical composition for treating and/or preventing age-relatedconditions, such as muscular atrophy.

In one embodiment, the present invention provides a pharmaceuticalcomposition (e.g., in an intra-muscular administering form) comprising(e.g., as a sole pharmaceutically active ingredient) a fusionpolypeptide (e.g., glycosylated or non-glycosylated) that comprisessKlotho of alpha Klotho protein with signal peptide (SEQ ID NO:44 or SEQID NO:45); and a linker comprising SEQ ID NO:11; and uses of thepharmaceutical composition for treating and/or preventing age-relatedconditions, such as muscular atrophy. In another embodiment, the presentinvention provides a pharmaceutical composition (e.g., in anintra-muscular administering form) comprising (e.g., as a solepharmaceutically active ingredient) a fusion polypeptide (e.g.,glycosylated or non-glycosylated) comprising sKlotho of alpha Klothoprotein without signal peptide (SEQ ID NO:7); and a linker comprisingSEQ ID NO:11; and uses of the pharmaceutical composition for treatingand/or preventing age-related conditions, such as muscular atrophy.

In one embodiment, the present invention provides a pharmaceuticalscomposition comprising (e.g., as a sole pharmaceutically activeingredient) a fusion polypeptide (e.g., glycosylated ornon-glycosylated) that comprises a human FGF protein or an activefragment thereof (e.g., without the signal peptide); and a linkercomprising SEQ ID NO:11.

Pharmaceutical compositions comprising the fusion proteins of theinvention and their uses for treating or preventing age-relatedconditions (e.g., muscle atrophy) or metabolic disorders (e.g., diabete)are also encompassed by the present invention.

In one embodiment, the present invention provides a fusion polypeptidethat is at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 96%, at least97%, at least 98%, at least 99% identical to SEQ ID NO:19. In anotherembodiment, the present invention provides a fusion polypeptide that isat least 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 96%, at least 97%, atleast 98%, at least 99% identical to SEQ ID NO: 20.

In one embodiment, the present invention provides a fusion polypeptidethat is at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 96%, at least97%, at least 98%, at least 99% identical to SEQ ID NO: 40. In anotherembodiment, the present invention provides a fusion polypeptide that isat least 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 96%, at least 97%, atleast 98%, at least 99% identical to SEQ ID NO: 41.

In one embodiment, the present invention provides a fusion polypeptidecomprising a sKlotho of beta Klotho protein with signal peptide fused(directly or indirectly via a linker) to FGF-19 or an active fragmentthereof. In another embodiment, the present invention provides a fusionpolypeptide comprising a sKlotho of beta Klotho protein without signalpeptide fused (directly or indirectly via a linker) to FGF-19 or anactive fragment thereof. In another embodiment, the present inventionprovides a fusion polypeptide comprising a sKlotho of beta Klothoprotein with signal peptide fused (directly or indirectly via a linker)to FGF-21 or an active fragment thereof. In another embodiment, thepresent invention provides a fusion polypeptide comprising a sKlotho ofbeta Klotho protein without signal peptide fused (directly or indirectlyvia a linker) to FGF-21 or an active fragment thereof.

The invention provides nucleic acid sequences encoding any of the Klothofusion polypeptides described herein and host cells containing thenucleic acids.

The invention also provides composition having any of the Klotho fusionpolypeptides contemplated herein. The compositions of the invention canfurther include heparin.

The invention also provides a method for treating or preventing anage-related condition in an individual. An individual (e.g, human) isadministered a therapeutically effective dose of a pharmaceuticalcomposition containing a Klotho fusion polypeptide, having at least oneextracellular subdomain of a Klotho protein (e.g., alpha Klotho protein)and a fibroblast growth factor or an active fragment thereof so as totreat or prevent the age-related condition. In particular, the inventionprovides a method of treating or preventing muscle wasting comprisingadministering to an individual (e.g., human) an therapeuticallyeffective amount of a fusion polypeptide having at least oneextracellular subdomain of an alpha Klotho protein and a fibroblastgrowth factor (or an active fragment thereof).

Additionally, the invention provides a method for treating or preventinga metabolic disorder in an individual. An individual is administered atherapeutically effective dose of a pharmaceutical compositioncontaining a fusion polypeptide of the invention, having at least oneextracellular subdomain of a Klotho protein and a fibroblast growthfactor (or an active fragment thereof) so as to treat the metabolicdisorder. In particular, a fusion polypeptide of the invention having atleast one extracellular subdomain of a beta-Klotho protein and afibroblast growth factor 21 is useful for treating a metabolic disorder.

Klotho-FGF23 fusion polypeptides of the invention can be used fortreating or preventing hyperphosphatemia or calcinosis in an individual.A pharmacologically effective dose of a pharmaceutical compositioncontaining the Klotho fusion polypeptide of the invention, having atleast one extracellular subdomain of a Klotho protein and a fibroblastgrowth factor, is administered to treat or prevent hyperphosphatemia orcalcinosis. In particular, a Klotho fusion polypeptide of the inventionhaving at least one extracellular subdomain of an alpha Klotho proteinand a fibroblast growth factor 23 is useful for treatinghyperphosphatemia or calcinosis.

Klotho-FGF23 fusion polypeptides of the invention can be used fortreating or preventing chronic renal disease or chronic renal failure inan individual. A therapeutically effective dose of a pharmaceuticalcomposition containing the Klotho fusion polypeptide of the invention,having at least one extracellular subdomain of a Klotho protein (e.g.,alpha Klotho protein) and a fibroblast growth factor, is administered totreat or prevent chronic renal disease or chronic renal failure.

Klotho-FGF23 fusion polypeptides of the invention can be used fortreating or preventing cancer (e.g., breast cancer) in an individual. Atherapeutically effective dose of a pharmaceutical compositioncontaining the Klotho fusion polypeptide of the invention, having atleast one extracellular subdomain of a Klotho protein (e.g., alphaKlotho protein) and a fibroblast growth factor, is administered to treator prevent cancer or breast cancer.

The present invention provides fusion polypeptides comprising at leastone extracellular subdomain of Klotho protein and a FGF or an activefragment thereof for use in medicine. In one embodiment, the presentinvention provides fusion polypeptides comprising at least oneextracellular subdomain of Klotho protein and a FGF or an activefragment thereof for use in treating or preventing muscle atrophy. Thepresent invention also provides a method of treating or preventing anage related condition (e.g., muscle atrophy) comprising administering toan individual in need thereof a therapeutically effective dose of apharmaceutical composition comprising a soluble Klotho protein.

The invention also includes kits for treating or preventing anage-related disorder or metabolic disorder in an individual. The kitincludes instructions for use and a purified Klotho fusion polypeptidehaving at least one extracellular subdomain of a Klotho protein and afibroblast growth factor.

The invention also provides a kit for producing a Klotho fusionpolypeptide of the invention. The kit of the invention includesinstructions for use and a nucleic acid encoding a Klotho fusionpolypeptide, having at least one extracellular subdomain of Klothoprotein and a fibroblast growth factor.

3. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates several different embodiments of the Klotho fusionpolypeptides of the invention. The represented fusion polypeptidesinclude one or more Klotho extracellular subdomains operatively linkedto a fibroblast growth factor. Polypeptides containing one or moreKlotho extracellular subdomains include, for example, an extracellulardomain of Klotho (e.g., a.a. 1 to 982 of human Klotho), or an activefragment of Klotho.

FIG. 2 illustrates the amino acid and nucleic acid sequences of severalKlotho fusion polypeptides of the invention and components thereof(e.g., Klotho extracellular domain, FGF).

FIGS. 3A-3C depict protein expression of an sKlotho-FGF23 fusionprotein. FIG. 3A shows that sKlotho-FGF23 fusion protein was detected inconditioned media by Western blotting with anti-FGF23 antibodies. FIG.3B shows that sKlotho-FGF23 fusion protein was detected in conditionedmedia by SDS-PAGE and Coomassie blue staining FIG. 3C shows a highlypurified sKlotho-FGF23-6×His fusion protein, analyzed by SDS-PAGE andCoomassie blue staining.

FIG. 4 illustrates the results of an Egr-1 luciferase assay comparingthe activation level of Egr-1 in cells treated with conditioned mediacontaining either a Klotho fusion polypeptide, a FGF 23 polypeptideonly, a soluble Klotho (sKlotho) polypeptide only, and a soluble Klothopolypeptide in combination with a FGF 23 polypeptide in the absence orpresence of heparin (20 μg ml).

FIGS. 5A-5B depict the results of an Egr-1 luciferase assay comparingthe activation level of Egr-1 in cells treated with purified Klothofusion polypeptide, FGF 23 polypeptide, or soluble Klotho polypeptide inthe absence or presence of heparin. FIG. 5A shows an the results of anexperiment comparing the activation level of Egr-1 in cells treated withFGF 23 alone, sKlotho-His (10 nM or 20 nM) and a combination of FGF 23and sKlotho-His (10 nM or 20 nM) in the absence or presence of heparin(20 μg/ml). FIG. 5B shows Egr-1 luciferase reporter activity in cellstreated with sKlotho-FGF23-His fusion (0 nM, 0.6 nM, 1.21 nM, 2.41 nM,4.83 nM, 9.65 nM, and 19.3 nM).

FIGS. 6A-6B illustrate the effect of treatment with a purified sKlothofusion polypeptide on C2C12 muscle cells. FIG. 6A shows measurements ofmyotube diameter in C2C12 muscle cells treated with either IGF-1 (10nM), FGF2 (20 ng ml), or a purified Klotho fusion polypeptide (20 nM),in the absence or presence of dexamethasone (100 μM). FIG. 6B shows thephosphorylation of signaling pathway proteins in C2C12 muscle cells byIGF-1 (10 nM), FGF2 (20 ng ml), or a purified Klotho fusion polypeptide(20 nM), in the absence or presence of rapamycin (40 nM).

4. DETAILED DESCRIPTION

The present invention is directed to methods, kits and compositions forpreventing or treating age-related conditions and metabolic disorders.The fusion polypeptides of the invention include a Klotho protein oractive fragment thereof. In some embodiments, the fusion polypeptides ofthe invention include a Klotho protein or an active fragment thereofoperatively linked to a fibroblast growth factor polypeptide or activefragment thereof. The Klotho fusion proteins or sKlotho of the presentinvention are useful in the treatment and prevention of a variety ofage-related conditions including sarcopenia, skin atrophy, musclewasting, brain atrophy, atherosclerosis, arteriosclerosis, pulmonaryemphysema, osteoporosis, osteoarthritis, immunologic incompetence, highblood pressure, dementia, Huntington's disease, Alzheimer's disease,cataracts, age-related macular degeneration, prostate cancer, stroke,diminished life expectancy, memory loss, wrinkles, impaired kidneyfunction, and age-related hearing loss; and metabolic disordersincluding Type II Diabetes, Metabolic Syndrome, hyperglycemia, andobesity.

The present invention, is based at least in part, on the finding thatdespite the physical constraints (e.g., large size of both the Klothoand FGF polypeptides) the Klotho-FGF fusion polypeptides are highlyeffective in activating an FGF receptor. This finding is unexpectedgiven that fusion of these two proteins would likely interfere with theheterodimerization and thus the activities of the proteins; e.g., thebinding domains of the proteins may be perturbed by the fusion or theproteins may be misoriented spatially if put together in a “cis”formation.

The Klotho-FGF fusion polypeptides described herein are advantageousbecause they allow the administration of a single therapeutic proteinthat has enhanced activity compared to Klotho or FGF administered aloneor together as separate polypeptides. The use of Klotho and FGF as asingle fusion polypeptide rather than as two separate polypeptides(i.e., a Klotho polypeptide and a separate FGF polypeptide) is moreeffective at activating the FGF receptor.

DEFINITIONS

“Klotho polypeptide”, “Klotho protein”, or “Klotho” as used herein,includes active fragments, derivatives, mimetics, variants andchemically modified compounds or hybrids thereof of wild-type “Klotho”.A Klotho active fragment has the ability to bind to an FGF polypeptide.Generally, a Klotho active polypeptide contains at least a Klothosubdomain (e.g., KL-D1 and KL-D2). Wild-type Klotho has the amino acidsequence as is found in nature. Exemplary Klotho polypeptides suitablefor use with the present invention include alpha-Klotho (SEQ ID NO: 2)and beta-Klotho (SEQ ID NO: 4). Nucleotide and amino acid sequences ofthe alpha-Klotho and beta-Klotho are found in the GenBank database atAccession No. NM_(—)004795; NP_(—)004786 and NM_(—)175737; NP_(—)783864,respectively. Klotho polypeptides include those described in U.S. Pat.No. 6,579,850, the content of which is herein incorporated by referencein its entirety. The Klotho polypeptides include those from otherspecies besides humans, including alpha-Klotho from mouse(NP_(—)038851), rat (NP_(—)112626), rabbit (NP_(—)001075692) andbeta-Klotho from mouse (NP_(—)112457). Species predicted to havealpha-Klotho include chimpanzee (XP_(—)522655), macaque(XP_(—)001101127), horse (XP_(—)001495662), cow (XP_(—)001252500),platypus (XP_(—)001510981), and chicken (XP_(—)417105). Speciespredicted to have beta-Klotho include chimpanzee (XP_(—)526550), macaque(XP_(—)001091413), horse (XP_(—)001495248), dog (XP_(—)536257), rat(XP_(—)001078178), platypus (XP_(—)001512722), and chicken(XP_(—)423224). The Klotho polypeptides have an amino acid sequence thatis substantially identical to the amino acid sequence of SEQ ID NO: 2 orSEQ ID NO:4; i.e., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99% or more identical at the amino acid sequences of SEQ ID NO:2 or SEQID NO:4, or active fragment thereof.

“Fusion polypeptide” or “fusion protein”, as used herein, shall mean apolypeptide comprising two or more different polypeptides or activefragments thereof that are not naturally present in the samepolypeptide. In some embodiments, the two or more different polypeptidesare operatively linked together covalently, e.g., chemically linked orfused in frame by a peptide bond. As used herein a “Klotho fusionpolypeptide” is a fusion polypeptide which includes an amino acidsequence from a Klotho polypeptide or active fragment thereof.

“Fibroblast growth factor” and “FGF” are used interchangeably herein andshall refer to polypeptides that regulate cell proliferation, migration,differentiation, homeostasis, tissue repair and response to injury in ananimal, including a human subject. FGFs have the ability to bind to afibroblast growth factor receptor and regulate its activity, includingautophosphorylation of FGFR, phosphorylation of FRS2 (FGF receptorsubstrate 2) and ERK1/2 (extracellular signal-regulated protein kinase½), and activating Egr-1 (early growth response-1). The term “FGF”includes active fragments, derivatives, mimetics, variants andchemically modified compounds or hybrids thereof of wild-type “FGF”,e.g., as known in the art and as described in U.S. Pat. No. 7,223,563and U.S. Pat. No. 7,259,248, the contents of which are incorporated byreference in their entirety. Wild-type FGF has an amino acid sequence asis found in nature. Exemplary fibroblast growth factors suitable for usewith the present invention include fibroblast growth factor-19 (FGF19;SEQ ID NO: 31), fibroblast growth factor-21 (FGF21; SEQ ID NO: 33), andfibroblast growth factor-23 (FGF23; SEQ ID NO: 35). The FGF polypeptidesinclude those from other species besides humans, including murine FGFs.Generally, FGF polypeptides have an amino acid sequence that issubstantially identical to the amino acid sequence of SEQ ID NO: 31, SEQID NO:33 or SEQ ID NO:35; i.e., having an amino acid sequence is whichis at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or moreidentical to the amino acid sequences of SEQ ID NO: 31 SEQ ID NO:33 orSEQ ID NO:35, or active fragments thereof.

The term “FGF”, includes active fragments of the full-lengthpolypeptide. Active FGF fragments that are able to bind to theircorresponding FGF receptors are known in the art and also contemplatedfor use in the present invention. One skilled in the art wouldappreciate, based on the sequences disclosed herein, that overlappingfragments of the FGFs can be generated using standard recombinanttechnology, for example, that described in Sambrook et al. (1989,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, New York) and Ausubel et al. (1997, Current Protocols inMolecular Biology, Green & Wiley, New York). One skilled in the artwould appreciate, based on the disclosure presented herein, that thebiological activity of FGF fragments could be tested by methods wellknown in the art and described herein, including binding to the FGFreceptor. Similarly, cell culture models which possess the necessary FGFsignal transduction machinery (i.e. FGF receptor) may be transfectedwith FGF fragments and subsequently tested for alterations in FGFsignaling, relative to wild type FGF.

FGFs are grouped into seven subfamilies based on the homology of the FGFcore homology domain (approximately 120 amino acids long), which isflanked by N- and C-terminal sequences that are highly variable in bothlength and primary sequence, particularly among different FGFsubfamilies (Goetz et al., Molecular and Cellular Biology, 2007, Vol 27,3417-3428). An FGF active polypeptide generally contains at least an FGFcore homology domain. In some embodiments, an FGF active polypeptide maycontain, in addition to an FGF core homology domain, flanking sequenceswhich may confer additional specificity in binding FGF receptors. FGF19,FGF21, and FGF23 are grouped in the FGF19 subfamily because the coreregion of these ligands share high sequence identity relative to otherFGFs (FGF19 v. FGF21: 38% identity; FGF19 v. FGF23: 36% identity). FGF19subfamily members act analogously to signaling molecules of theendocrine system and regulate diverse physiological processes uncommonto classical FGFs (e.g., FGF19: energy and bile acid homeostasis; FGF21:glucose and lipid metabolism; and FGF 23: phosphate and vitamin Dhomeostasis).

“Fibroblast growth factor receptor” and “FGFR” as used herein refer toany one of FGFRs 1-4 known in the art, or splice variants thereof (e.g.,FGFR1c). Exemplary fibroblast growth factor receptors suitable for usewith the present invention include fibroblast growth factor receptor-19(e.g., FGFR4-beta Klotho), fibroblast growth factor receptor-21 (e.g.,FGFR1c-alpha Klotho), and fibroblast growth factor receptor-23 (e.g.,FGFR1c-alpha Klotho, FGFR3-alpha Klotho, FGFR4-alpha Klotho).

“Extracellular domain”, as used herein, refers to the fragment of atransmembrane protein existing outside of a cell (e.g., not includingthe intracellular or transmembrane region). The “extracellular domain ofthe Klotho protein”, “soluble Klotho”, or “sKlotho” (e.g., SEQ ID NO: 7;SEQ ID NO: 39), refers to an extracellular domain of the Klothopolypeptide that is capable of binding a fibroblast growth factor,and/or capable of enabling the binding of a fibroblast growth factor toa fibroblast growth factor receptor by binding to the fibroblast growthfactor. The Klotho extracellular domain corresponds to amino acidresidues 28-982 of the full length alpha Klotho sequence (SEQ ID NO: 2)and to amino acid residues 52-997 of the full length beta Klothosequence (SEQ ID NO:4).

“Extracellular subdomain of Klotho protein” and “extracellular subdomainof Klotho protein” are used interchangeably herein and shall refer to aregion in the extracellular domain of the Klotho polypeptide that iscapable of binding a fibroblast growth factor, and/or is capable ofenabling the binding of a fibroblast growth factor to a fibroblastgrowth factor receptor by binding to the fibroblast growth factor. TheKlotho extracellular domain has two homologous subdomains that arerepeated, i.e., KL-D1 (SEQ ID NO: 5) and KL-D2 (SEQ ID NO: 6). KL-D1 andKL-D2 correspond respectively to amino acid residues 58-506 and 517-953of the full length alpha Klotho polypeptide (SEQ ID NO: 2) andrespectively to amino acid residues 77-508 and 571-967 of the fulllength beta Klotho polypeptide (SEQ ID NO:4) and are suitable for usewith the present invention. Generally, a polypeptide that contains atleast one Klotho subdomain is a Klotho active polypeptide. The Klothoextracellular subdomain for use with the polypeptide of the inventionmay be an alpha Klotho or beta Klotho KL-D1 domain with an amino acidsequence that is substantially identical to the amino acid sequence ofSEQ ID NO: 5 or SEQ ID NO: 37, respectively. Further, the Klotho KL-D1domain may have an amino acid sequence that is at least 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acidsequence of SEQ ID NO: 5 or SEQ ID NO: 37. The Klotho extracellularsubdomain may also be an alpha or beta Klotho polypeptide KL-D2 domainthat is substantially identical to the amino acid sequence of SEQ ID NO:6 or SEQ ID NO: 38, respectively. In a further embodiment, the KL-D2domain has an amino acid sequence that is at least at least 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the aminoacid sequence of SEQ ID NO: 6 or SEQ ID NO: 38.

“Signal peptide”, as used herein, shall mean a peptide chain (3-60 aminoacids long) that directs the post-translational transport of a proteinto the endoplasmic reticulum and may be cleaved off. Exemplary signalpeptides suitable for use with the present invention include the Klothosignal peptide (SEQ ID NO:19) and the IgG signal peptide (SEQ ID NO:20).

“Linker”, as used herein, shall mean a functional group (e.g., chemicalor polypeptide) that covalently attaches two or more polypeptides ornucleic acids so that they are connected with one another. As usedherein, a “peptide linker” refers to one or more amino acids used tocouple two proteins together (e.g., to couple the extracellular domainof Klotho and fibroblast growth factor-23). Peptide linkers suitable foruse with the present invention include, but are not limited to,polypeptides with amino acid sequences represented by SEQ ID NO:8, SEQID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18.

“Operatively linked”, as used herein, shall mean the linking of two ormore biomolecules so that the biological functions, activities, and/orstructure associated with the biomolecules are at least retained. Inreference to polypeptides, the term means that the linking of two ormore polypeptides results in a fusion polypeptide that retains at leastsome of the respective individual activities of each polypeptidecomponent. The two or more polypeptides may be linked directly or via alinker. In reference to nucleic acids, the term means that a firstpolynucleotide is positioned adjacent to a second polynucleotide thatdirects transcription of the first polynucleotide when appropriatemolecules (e.g., transcriptional activator proteins) are bound to thesecond polynucleotide.

“Specifically binds”, as used herein, shall refer to the ability of afirst molecule to bind to a target molecule out of many, different typesof molecules to which it may be exposed because of the ability of thefirst molecule to adopt a particular structure conducive to formingnoncovalent interactions between itself and the other target molecule.The first molecule binds to the target forming a stable complex whilethere is substantially less recognition, contact, or complex formationof the first molecule with any other non-specific molecules.

“Polypeptide variant” or “protein variant”, as used herein, refers topolypeptides in which one or more amino acids have been substituted bydifferent amino acids from a reference sequence. It is well understoodin the art that some amino acids may be substituted by others withbroadly similar properties without changing the nature of the activityof the polypeptide (conservative substitutions) as describedhereinafter. These terms also encompass polypeptides in which one ormore amino acids have been added or deleted, or replaced with differentamino acids, e.g., protein isoforms. An exemplary variant of fibroblastgrowth factor-23 suitable for use with the present invention is thefibroblast growth factor-23 variant (R179Q).

“Pharmaceutical composition”, as used herein, shall mean a compositioncontaining a compound (e.g., a fusion polypeptide of the invention) thatmay be administered to treat or prevent a disease or disorder in anindividual.

“Individual” or “subject”, as used herein, shall refer to a mammal,including, but not limited to, a human or non-human mammal, such as abovine, equine, canine, ovine, or feline.

“Treat”, as used herein, shall mean decrease, suppress, attenuate,diminish, arrest, or stabilize the development or progression of adisease. In the context of the invention, the administration of thepolypeptides of the invention may be used to treat age-relatedconditions, including sarcopenia, skin atrophy, muscle wasting, brainatrophy, atherosclerosis, arteriosclerosis, pulmonary emphysema,osteoporosis, osteoarthritis, immunologic incompetence, high bloodpressure, dementia, Huntington's disease, Alzheimer's disease,cataracts, age-related macular degeneration, prostate cancer, stroke,diminished life expectancy, memory loss, wrinkles, impaired kidneyfunction, and age-related hearing loss; and metabolic disorders,including Type II Diabetes, Metabolic Syndrome, hyperglycemia, andobesity.

“Prevent”, as used herein, shall refer to a decrease in the occurrenceof a disorder or decrease in the risk of acquiring a disorder or itsassociated symptoms in a subject. In the context of the invention, theadministration of the polypeptides of the invention may be used toprevent age-related conditions, including sarcopenia, skin atrophy,muscle wasting, brain atrophy, atherosclerosis, arteriosclerosis,pulmonary emphysema, osteoporosis, osteoarthritis, immunologicincompetence, high blood pressure, dementia, Huntington's disease,Alzheimer's disease, cataracts, age-related macular degeneration,prostate cancer, stroke, diminished life expectancy, memory loss,wrinkles, impaired kidney function, and age-related hearing loss; andmetabolic disorders, including Type II Diabetes, Metabolic Syndrome,hyperglycemia, and obesity. The prevention may be complete, e.g., thetotal absence of an age-related condition or metabolic disorder. Theprevention may also be partial, such that the likelihood of theoccurrence of the age-related condition or metabolic disorder in asubject is less likely to occur than had the subject not received thepresent invention.

“Disease”, as used herein, shall mean any condition or disorder thatdamages or interferes with the normal function of a cell, tissue, ororgan.

“Age-related condition”, as used herein, shall mean any disease ordisorder whose incidence in a population or severity in an individualcorrelates with the progression of age.

In one embodiment, the age-related condition is a disease or disorderwhose incidence is at least 1.5 fold higher among human individualsgreater than 60 years of age relative to human individuals between theages of 30-40 and in a selected population of greater than 100,000individuals. Age-related conditions relevant to the present inventioninclude, but are not limited to, sarcopenia, skin atrophy, musclewasting, brain atrophy, atherosclerosis, arteriosclerosis, pulmonaryemphysema, osteoporosis, osteoarthritis, immunologic incompetence, highblood pressure, dementia, Huntington's disease, Alzheimer's disease,cataracts, age-related macular degeneration, prostate cancer, stroke,diminished life expectancy, memory loss, wrinkles, impaired kidneyfunction, and age-related hearing loss.

“Metabolic disorder”, as used herein, shall mean any disease or disorderthat damages or interferes with normal function in a cell, tissue, ororgan by affecting the production of energy in cells or the accumulationof toxins in a cell, tissue, organ, or individual. Metabolic disordersrelevant to the present invention include, but are not limited to, TypeII Diabetes, Metabolic Syndrome, hyperglycemia, and obesity.

An “effective dose” or “effective amount” is an amount sufficient toeffect a beneficial or desired clinical result. In the context of theinvention, it is an amount of a Klotho fusion polypeptide or sKlothoeffective to produce the intended pharmacological, therapeutic orpreventive result. A therapeutically effective dose results in theprevention or amelioration of the disorder or one or more symptoms ofthe disorder, (e.g., an age-related condition or metabolic disorder).Therapeutically effective doses will vary depending upon the subject anddisease condition being treated, the weight and age of the subject, theseverity of the disease condition, the manner of administration and thelike which can be readily be determined by one of ordinary skill in theart.

“Klotho nucleic acid molecule”, as used herein is a gene encoding aKlotho protein. An exemplary human Klotho gene is provided at GenBankAccession No. NM_(—)004795 (SEQ ID NO:1).

“Fragment”, as used herein, refers to a portion of a polypeptide ornucleic acid molecule. This portion contains, preferably, at least 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the entire length ofthe reference nucleic acid molecule or polypeptide. A fragment maycontain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500,600, 700, 800, 900, 1000 or up to 3000 nucleotides or amino acids.

The term “substantially identical” refers to a polypeptide or nucleicacid molecule exhibiting at least 50% identity to a reference amino acidsequence (for example, any one of the amino acid sequences describedherein) or nucleic acid sequence (for example, any one of the nucleicacid sequences described herein). Preferably, such a sequence is atleast 60%, 70%, 75%, 80% or 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more identical at the amino acid level or nucleic acid tothe sequence used for comparison.

The present invention is directed to methods, kits and compositions forpreventing or treating age-related conditions and metabolic disorders.The invention provides a fusion polypeptide having at least oneextracellular subdomain of a Klotho protein. In some embodiments, thefusion polypeptides further comprise a fibroblast growth factor or anactive fragment thereof. The Klotho extracellular domain may be derivedfrom either the alpha or beta Klotho isoforms. Further, although the FGFcomponent of the Klotho fusion polypeptide is described primarily withreference to fibroblast growth factor-19, fibroblast growth factor-21and fibroblast growth factor-23, it is contemplated that any of thetwenty-three known FGFs or an active fragment thereof can be used inpracticing the invention.

The extracellular domain of the Klotho protein can include one or bothof the KL-D1 and KL-D2 domains of a Klotho protein. In some embodiments,the Klotho fusion polypeptide has at least two extracellular subdomainsof a Klotho protein. For example, the two extracellular subdomains canbe two KL-D1 domains in tandem repeats, two KL-D2 domains in tandemrepeats, or one KL-D1 domain and one KL-D2 domain.

The extracellular subdomain of a Klotho protein and the fibroblastgrowth factor (or an active fragment thereof) can be operatively linkedto one another in a variety of orientations and manners. For example,the extracellular subdomain of the Klotho protein can be operativelylinked to the N-terminus of the fibroblast growth factor oralternatively the fibroblast growth factor can be operatively linked tothe N-terminus of the at least one extracellular subdomain of the Klothoprotein.

The fusion polypeptide of the invention may include one or both of theKlotho extracellular domains, i.e., KL-D1 (SEQ ID NO: 5) and KL-D2 (SEQID NO: 6). KL-D1 and KL-D2 correspond respectively to amino acidresidues 58-506 and 517-953 of the full length alpha Klotho polypeptide(SEQ ID NO: 2) and to amino acid residues 77-508 and 571-967 of the fulllength beta Klotho polypeptide (SEQ ID NO:4) and are suitable for usewith the present invention. The Klotho fusion polypeptide may have aKL-D1 domain of an alpha Klotho polypeptide having an amino acidsequence that is substantially identical to the amino acid sequence ofSEQ ID NO: 5 or of a beta Klotho polypeptide having an amino acidsequence that is substantially identical to the amino acid sequence ofSEQ ID NO: 37. Specifically, the Klotho fusion polypeptide may have anamino acid sequence that is at least at least 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 5 or SEQ ID NO:37. The Klotho fusion polypeptide may have a KL-D2 domain of an alphaKlotho polypeptide with an amino acid sequence that is substantiallyidentical to the amino acid sequence of SEQ ID NO: 6 or of a beta Klothopolypeptide having an amino acid sequence that is substantiallyidentical to the amino acid sequence of SEQ ID NO: 38. Specifically, theKlotho fusion polypeptide may have an amino acid sequence that is atleast at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or moreidentical to SEQ ID NO: 6 or SEQ ID NO: 38, respectively.

In some embodiments, the Klotho fusion polypeptide of the invention issoluble and is capable of binding to an FGF receptor.

The Klotho fusion polypeptides of the invention can contain apolypeptide linker which connects the polypeptide having at least oneextracellular subdomain of a Klotho protein and the fibroblast growthfactor. Suitable linkers are well known in the art and generally containseveral Gly and several Ser residues, e.g., (Gly₄ Ser)₃ (SEQ ID NO:11),Gly₄ Ser polypeptide (SEQ ID NO: 12), Gly (SEQ ID NO: 13), Gly Gly (SEQID NO: 14), Gly Ser (SEQ ID NO: 15), Gly₂ Ser (SEQ ID NO: 16), Ala (SEQID NO: 17), and Ala Ala (SEQ ID NO: 18). In some embodiments, the linkerwill have at least 2 and up to about 30 repeats of an amino acidsequence represented by any one of SEQ ID NO: 12, SEQ ID NO: 13, SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18.

When a polypeptide linker is present in the Klotho fusion polypeptide ofthe invention, the polypeptide having at least one extracellularsubdomain of a Klotho protein may be connected by a peptide bond to theN-terminus of the linker polypeptide with the FGF connected by a peptidebond to the C-terminus of the polypeptide linker. Alternatively, the FGFmay be connected by a peptide bond to the N-terminus of the linkerpolypeptide with the polypeptide having at least one extracellularsubdomain of Klotho connected by a peptide bond to the C-terminus of thepolypeptide linker. A chemical linker can also be used to link the twopolypeptides.

The Klotho fusion polypeptide of the invention may include a signalpeptide. Exemplary signal peptides for use with the Klotho fusionpolypeptide include, but are not limited to the Klotho signal peptide(SEQ ID NO: 8) and the IgG signal peptide (SEQ ID NO: 9).

4.1. Klotho and Fibroblast Growth Factor Polypeptides

The Klotho fusion polypeptides of the invention are expected to exhibitbiological activities comparable to FGF in nature, such as binding to anFGF receptor and inducing the phosphorylation of an FGF receptor, FRS2(FGF receptor substrate 2) and ERK1/2 (extracellular signal-regulatedprotein kinase ½) and activating Egr-1 (early growth response-1) gene.FGF is a secreted peptide growth factor that binds the FGF receptor. Theamino acid and nucleic acid sequences of FGF are readily available tothose of skill in the art. For example, exemplary nucleotide sequencesfor FGF19, FGF21, and FGF23 can be found in the GenBank database atAccession numbers: NM_(—)005117, NM_(—)019113, and NM_(—)020638,respectively, and herein as SEQ ID NOs: 30, 32, and 34, respectively.Exemplary amino sequences for FGF19, FGF21, and FGF23 can be found inthe GenBank database at Accession numbers: NP_(—)005108, NP_(—)061986,and NP_(—)065689, respectively, and herein as SEQ ID NOs: 31, 35, and35, respectively. Additionally, FGF may include one or more alterationswhich aid in the expression of the protein, e.g., the FGF23 (R179Q)variant (SEQ ID NO: 36).

The Klotho protein is a 130 kDa single pass type I transmembrane proteinwith an extracellular domain and a short cytoplasmic domain. The aminoacid and nucleic acid sequences of Klotho are readily available to thoseof skill in the art. For example, exemplary nucleotide sequences foralpha-Klotho and beta-Klotho can be found in the GenBank database atAccession numbers: NM_(—)004795 and NM_(—)175737, respectively, andherein as SEQ ID NOs: 7 and 8, respectively. Exemplary amino acidsequences for alpha-Klotho and beta-Klotho can be found in the GenBankdatabase at Accession numbers: NP_(—)004786 and NP_(—)783864,respectively, and herein as SEQ ID NOs: 2 and 4, respectively.

The Klotho fusion polypeptide of the invention can bind to a fibroblastgrowth factor receptor and has an alpha-Klotho or beta-Klothoextracellular domain operatively linked to either fibroblast growthfactor-19 (SEQ ID NO: 31), fibroblast growth factor-21 (SEQ ID NO: 33),fibroblast growth factor-23 (SEQ ID NO: 35), or variants thereof (whichinclude fibroblast growth factor-23 variant (R179Q) (SEQ ID NO: 36)).

Specifically, the Klotho fusion polypeptide of the invention may includean alpha-Klotho (SEQ ID NO: 2) which is operatively coupled tofibroblast growth factor-23 (SEQ ID NO: 35) or fibroblast growthfactor-23 variant (R179Q) (SEQ ID NO: 36). Additionally, the Klothofusion polypeptide of the invention may have beta-Klotho (SEQ ID NO: 4),which is operatively coupled to fibroblast growth factor-19 (SEQ ID NO:31). The Klotho fusion polypeptide of the invention may include abeta-Klotho (SEQ ID NO: 4), which is operatively coupled to fibroblastgrowth factor-21 (SEQ ID NO: 33).

The invention includes homologs of the various Klotho and FGF genes andproteins encoded by those genes. A “homolog,” in reference to a generefers to a nucleotide sequence that is substantially identical over atleast part of the gene or to its complementary strand or a part thereof,provided that the nucleotide sequence encodes a protein that hassubstantially the same activity/function as the protein encoded by thegene which it is a homolog of Homologs of the genes described herein canbe identified by percent identity between amino acid or nucleotidesequences for putative homologs and the sequences for the genes orproteins encoded by them (e.g., nucleotide sequences for genes encodingKlotho and FGF or their complementary strands). Percent identity may bedetermined, for example, by visual inspection or by using variouscomputer programs known in the art or as described herein. Sequenceidentity is typically measured using sequence analysis software (forexample, Sequence Analysis Software Package of the Genetics ComputerGroup, University of Wisconsin Biotechnology Center, 1710 UniversityAvenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOXprograms). Such software matches identical or similar sequences byassigning degrees of homology to various substitutions, deletions,and/or other modifications. Conservative substitutions typically includesubstitutions within the following groups: glycine, alanine; valine,isoleucine, leucine; aspartic acid, glutamic acid, asparagine,glutamine; serine, threonine; lysine, arginine; and phenylalanine,tyrosine. In an exemplary approach to determining the degree ofidentity, a BLAST program may be used, with a probability score betweene⁻³ and e⁻¹⁰⁰ indicating a closely related sequence.

As used herein, the terms “homology” and “homologous” are not limited todesignate proteins having a theoretical common genetic ancestor, butincludes proteins which may be genetically unrelated that have,nonetheless, evolved to perform similar functions and/or have similarstructures. Functional homology to the various proteins described hereinalso encompasses proteins that have an activity of the correspondingprotein of which it is a homolog. For proteins to have functionalhomology, it is not required that they have significant identity intheir amino acid sequences, but, rather, proteins having functionalhomology are so defined by having similar or identical activities. Forexample, with respect to a Klotho molecule, the polypeptide should havethe functional characteristics of binding to an FGF polypeptide andenable the binding of the FGF to an FGFR. With respect to an FGFmolecule, the polypeptide should have the functional characteristics ofbinding to an FGFR and causing the activation of FGFR (e.g.,phosphorylation). Assays for assessing FGF binding to the FGF receptorand/or activation of the FGF signaling pathway are known in the art anddescribed herein (See Example 2). Assays for assessing Klotho activityare also known in the art and described herein (e.g., binding to a FGFpolypeptide). Proteins with structural homology are defined as havinganalogous tertiary (or quaternary) structure and do not necessarilyrequire amino acid identity or nucleic acid identity for the genesencoding them. In certain circumstances, structural homologs may includeproteins which maintain structural homology only at the active site orbinding site of the protein.

In addition to structural and functional homology, the present inventionfurther encompasses proteins having amino acid identity to the variousKlotho and FGF amino acid sequences described herein. To determine thepercent identity/homology of two amino acid sequences, the sequences arealigned for optimal comparison purposes (e.g., gaps can be introduced inthe amino acid sequence of one protein for optimal alignment with theamino acid sequence of another protein). The amino acid residues atcorresponding amino acid positions are then compared. When a position inone sequence is occupied by the same amino acid residue as thecorresponding position in the other, then the molecules are identical atthat position. The percent identity between the two sequences is afunction of the number of identical positions shared by the sequences(i.e., % identity=# of identical positions/total # of positionsmultiplied by 100).

The amino acid sequences of molecules of the invention described hereinhave an amino acid sequence which is at least about 60%, 70%, 80%, 90%,95%, 96%, 97%, 98%, 99% or more identical or homologous to an amino acidsequence described herein.

The nucleic acid sequences of molecules of the invention describedherein have a nucleotide sequence which hybridizes to or is at leastabout 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identical orhomologous to a nucleotide sequence described herein.

Nucleic acid molecules appropriate for use in the fusion polypeptides ofthe invention may have a Klotho or FGF nucleotide sequence whichhybridizes under stringent conditions to the complement of a nucleicacid molecule encoding Klotho or FGF, respectively. As used herein, theterm “hybridizes under stringent conditions” is intended to describeconditions for hybridization and washing under which nucleotidesequences at least about 70%, 80%, 85%, 90% or more homologous to eachother typically remain hybridized to each other. Such stringentconditions are known to those skilled in the art and can be found inAusubel et al. Current Protocols in Molecular Biology, WileyInterscience, New York (2001), 6.3.1-6.3.6. A specific, non-limitingexample of stringent hybridization conditions are hybridization in 6×sodium chloride/sodium citrate (SSC) at about 45° C., followed by one ormore washes in 0.2×SSC, 0.1% SDS at 50-65° C.

4.2. Klotho-FGF Fusion Polypeptides of the Invention

In some embodiments of the invention, a Klotho fusion polypeptide has apolypeptide chain having a first polypeptide sequence of a Klothopolypeptide or an active fragment thereof and a second polypeptidesequence encoding FGF or an active fragment thereof.

The invention includes fusion polypeptides which are at least about 95%or more homologous to an amino acid sequence presented in SEQ IDNO:19-28. The amino acid sequence of SEQ ID NO: 19 encodes a Klothofusion polypeptide having a Klotho extracellular domain N-terminallylinked to the FGF23 (R179Q) variant (SEQ ID NO: 36). The amino acidsequence of SEQ ID NO: 20 encodes a Klotho fusion polypeptide having anIgG signal peptide N-terminally linked to a Klotho extracellular domainlacking a signal peptide N-terminally linked to the FGF23 (R179Q)variant. The amino acid sequence of SEQ ID NO: 21 encodes a Klothofusion polypeptide having a KL-D1 extracellular subdomain N-terminallylinked to the FGF23 (R179Q) variant. The amino acid sequence of SEQ IDNO: 22 encodes a Klotho fusion polypeptide having a KL-D2 extracellularsubdomain N-terminally linked to the FGF23 (R179Q) variant. The aminoacid sequence of SEQ ID NO: 23 encodes a Klotho fusion polypeptidehaving two KL-D1 extracellular subdomains N-terminally linked to theFGF23 (R179Q) variant. The amino acid sequence of SEQ ID NO: 24 encodesa Klotho fusion polypeptide having two KL-D2 extracellular subdomainsN-terminally linked to the FGF23 (R179Q) variant. The amino acidsequence of SEQ ID NO: 25 encodes a Klotho fusion polypeptide having theFGF23 (R179Q) variant N-terminally linked to a Klotho extracellulardomain. The amino acid sequence of SEQ ID NO: 26 encodes a Klotho fusionpolypeptide having the FGF23 (R179Q) variant N-terminally linked to aKL-D1 extracellular subdomain. The amino acid sequence of SEQ ID NO: 27encodes a Klotho fusion polypeptide having the FGF23 (R179Q) variantN-terminally linked to a KL-D2 extracellular subdomain. The amino acidsequence of SEQ ID NO: 28 encodes a Klotho fusion polypeptide having theFGF23 (R179Q) variant N-terminally linked to two KL-D1 extracellularsubdomains. The amino acid sequence of SEQ ID NO: 29 encodes a Klothofusion polypeptide having the FGF23 (R179Q) variant N-terminally linkedto two KL-D2 extracellular subdomains.

The Klotho fusion polypeptide of the invention may include an amino acidsequence which is at least about 95% identical to the amino acidsequence set forth in SEQ ID NO:7. The amino acid sequence of SEQ ID NO:7 encodes a Klotho extracellular domain lacking a signal peptide.

The subject fusion proteins are described herein and can be made usingmethods known in the art. For example, the fusion polypeptides of theinvention may be constructed as described in U.S. Pat. No. 6,194,177.The use of Klotho polypeptides is described in U.S. Pat. No. 6,579,850.The use of FGF nucleic acid molecules is described in U.S. Pat. No.7,223,563.

In some embodiments, a nucleic acid molecule encoding the Klotho iscloned by PCR and ligated, in frame, with a nucleic acid moleculeencoding FGF. The nucleic acid encoding the Klotho-FGF fusionpolypeptide is operatively linked to a promoter to allow for expression.The nucleic acid molecule encoding the fusion polypeptide issubsequently transfected into a host cell for expression. The sequenceof the final construct can be confirmed by sequencing.

When preparing the fusion proteins of the present invention, a nucleicacid molecule encoding an extracellular subdomain of Klotho will befused in frame to the nucleic acid molecule encoding FGF. Expression ofthe resulting nucleic acid molecule results in the extracellularsubdomain of Klotho being fused N-terminal in relation to the FGFpolypeptide. Fusions are also possible in which the extracellularsubdomain of Klotho is fused C-terminal in relation to the FGFpolypeptide. Methods for making fusion proteins are well known in theart.

The fusion polypeptides of the invention have at least two polypeptidesthat are covalently linked, in which one polypeptide comes from oneprotein sequence or domain, e.g., Klotho, and the other polypeptidecomes from another protein sequence or domain, e.g., FGF. Klotho andFGF, of the fusion polypeptides of the invention, can be joined bymethods well known to those of skill in the art. These methods includeboth chemical and recombinant means.

Nucleic acids encoding the domains to be incorporated into the fusionpolypeptides of the invention can be obtained using routine techniquesin the field of recombinant genetics. Basic texts disclosing the generalmethods of use in this invention include Sambrook and Russell, MolecularCloning, A Laboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer andExpression: A Laboratory Manual (1990); and Current Protocols inMolecular Biology (Ausubel et al., eds., 1994-1999). In nucleic acidsencoding a Klotho fusion polypeptide of the invention, the nucleic acidsequence encoding alpha-Klotho or beta-Klotho, represented by SEQ ID NO:1 and SEQ ID NO: 3, respectively, may be used. In nucleic acids encodinga Klotho fusion polypeptide, the nucleic acid sequence encoding FGF19,FGF21, or FGF23, represented by SEQ ID NO: 30, SEQ ID NO: 32 and SEQ IDNO: 34, respectively, may be used. Nucleic acid sequences of moleculesof the invention described herein comprise a nucleotide sequence whichhybridizes to or is at least about 60%, 70%, 80%, 90%, 95%, 96%, 97%,98%, 99% or more identical or homologous to SEQ ID NO: 1, SEQ ID NO:3,SEQ ID NO: 30, SEQ ID NO: 32, or SEQ ID NO: 34.

Nucleic acid sequences that encode Klotho and FGF peptides can beobtained using any of a variety of methods. For example, the nucleicacid sequences encoding the polypeptides may be cloned from cDNA andgenomic DNA libraries by hybridization with probes, or isolated usingamplification techniques with oligonucleotide primers. More commonly,amplification techniques are used to amplify and isolate the Klotho andFGF sequences using a DNA or RNA template (see, e.g., Dieffenfach &Dveksler, PCR Primers: A Laboratory Manual (1995)). Alternatively,overlapping oligonucleotides can be produced synthetically and joined toproduce one or more of the domains. Nucleic acids encoding Klotho or FGFcan also be isolated from expression libraries using antibodies asprobes.

According to the present invention, Klotho and FGF can be linked eitherdirectly or via a covalent linker, including amino acid linkers, such asa polyglycine linker, or another type of chemical linker, including,carbohydrate linkers, lipid linkers, fatty acid linkers, polyetherlinkers, such as PEG, etc. (See for example, Hermanson, Bioconjugatetechniques (1996)). The polypeptides forming the fusion fusionpolypeptide are typically linked C-terminus to N-terminus, although theycan also be linked C-terminus to C-terminus, N-terminus to N-terminus,or N-terminus to C-terminus. One or more polypeptide domains may beinserted at an internal location within a fusion polypeptide of theinvention. The polypeptides of the fusion protein can be in any order.The fusion polypeptides may be produced by covalently linking a chain ofamino acids from one protein sequence, e.g., an extacellular subdomainof Klotho, to a chain of amino acids from another protein sequence,e.g., FGF, by preparing a recombinant polynucleotide contiguouslyencoding the fusion protein. The different chains of amino acids in afusion protein may be directly spliced together or may be indirectlyspliced together via a chemical linking group or an amino acid linkinggroup. The amino acid linking group can be about 200 amino acids or morein length, or generally 1 to 100 amino acids. In some embodiments,proline residues are incorporated into the linker to prevent theformation of significant secondary structural elements by the linker.Linkers can often be flexible amino acid subsequences that aresynthesized as part of a recombinant fusion protein. Such flexiblelinkers are known to persons of skill in the art.

According to the present invention, the amino acid sequence of anextracellular subdomain of Klotho or a fragment thereof may be linked tothe FGF via a peptide linker. Exemplary peptide linkers are well knownin the art and described herein. For example, peptide linkers generallyinclude several Gly and several Ser residues, such as: (Gly₄ Ser)₃ (SEQID NO: 11), Gly₄ Ser polypeptide (SEQ ID NO: 12), Gly (SEQ ID NO: 13),Gly Gly (SEQ ID NO: 14), Gly Ser (SEQ ID NO: 15), Gly₂ Ser (SEQ ID NO:16), Ala (SEQ ID NO: 17), and Ala Ala (SEQ ID NO: 18). Specifically, apeptide linker for use in a fusion protein of the invention may act as aflexible hinge.

The signal sequence of Klotho or FGF may be excluded prior toincorporation of Klotho into a fusion protein of the invention. Thesignal sequence for Klotho or FGF of the fusion protein may be included,e.g., the polypeptide represented by SEQ ID NO: 19. However, suchsequences may also be omitted and replaced with the signal sequence of adifferent protein, e.g., the IgG signal sequence (SEQ ID NO: 9).Generally, the pharmaceutical compositions of the invention will containthe mature form of Klotho and FGF.

Generally, introns are excluded from either one or both the Klotho orthe FGF moieties prior to incorporation into a fusion polypeptide.

The fusion polypeptides of the invention may include one or morepolymers covalently attached to one or more reactive amino acid sidechains. By way of example, not limitation, such polymers includepolyethylene glycol (PEG), which can be attached to one or more freecysteine sulfhydryl residues, thereby blocking the formation ofdisulfide bonds and aggregation when the protein is exposed to oxidizingconditions. In addition, PEGylation of the fusion polypeptides of theinvention is expected to provide such improved properties as increasedhalf-life, solubility, and protease resistance. The fusion polypeptidesof the invention may alternatively be modified by the covalent additionof polymers to free amino groups such as the lysine epsilon or theN-terminal amino group. Preferred cysteines and lysines for covalentmodification will be those not involved in receptor binding, heparinbinding, or in proper protein folding. It will be apparent to oneskilled in the art that the methods for assaying the biochemical and/orbiological activity of the fusion polypeptides may be employed in orderto determine if modification of a particular amino acid residue affectsthe activity of the protein as desired. Other similar suitablemodifications are contemplated and known in the art.

The invention is also directed to the expression of a fusion polypeptidethat is at least about 95% or more homologous to an amino acid sequencepresented in SEQ ID NO:19-28.

4.3. Expression of Fusion Polypeptides of the Invention

In order to express the fusion protein of the invention, DNA moleculesobtained by any of the methods described herein or those that are knownin the art, can be inserted into appropriate expression vectors bytechniques well known in the art. For example, a double stranded cDNAcan be cloned into a suitable vector by homopolymeric tailing or byrestriction enzyme linking involving the use of synthetic DNA linkers orby blunt-ended ligation. DNA ligases are usually used to ligate the DNAmolecules and undesirable joining can be avoided by treatment withalkaline phosphatase.

Therefore, the invention includes vectors (e.g., recombinant plasmidsand bacteriophages) that include nucleic acid molecules (e.g., genes orrecombinant nucleic acid molecules encoding genes) as described herein.The term “recombinant vector” includes a vector (e.g., plasmid, phage,phasmid, virus, cosmid, fosmid, or other purified nucleic acid vector)that has been altered, modified or engineered such that it containsgreater, fewer or different nucleic acid sequences than those includedin the native or natural nucleic acid molecule from which therecombinant vector was derived. For example, a recombinant vector mayinclude a nucleotide sequence encoding a Klotho-FGF23 fusion operativelylinked to regulatory sequences, e.g., promoter sequences, terminatorsequences and/or artificial ribosome binding sites (RBSs), as definedherein. Recombinant vectors which allow for expression of the genes ornucleic acids included in them are referred to as “expression vectors.”

For eukaryotic hosts, different transcriptional and translationalregulatory sequences may be employed, depending on the nature of thehost. They may be derived from viral sources, such as adenovirus, bovinepapilloma virus, Simian virus or the like, where the regulatory signalsare associated with a particular gene which has a high level ofexpression. Examples include, but are not limited to, the TK promoter ofthe Herpes virus, the SV40 early promoter, the yeast ga14 gene promoter,etc. Transcriptional initiation regulatory signals may be selected whichallow for repression or activation, so that expression of the genes canbe modulated.

In some of the molecules of the invention described herein, one or moreDNA molecules having a nucleotide sequence encoding one or morepolypeptide chains of a fusion polypeptide are operatively linked to oneor more regulatory sequences, which are capable of integrating thedesired DNA molecule into a host cell. Cells which have been stablytransformed by the introduced DNA can be selected, for example, byintroducing one or more markers which allow for selection of host cellswhich contain the expression vector. A selectable marker gene can eitherbe linked directly to a nucleic acid sequence to be expressed, or beintroduced into the same cell by co-transfection. Additional elementsmay also be needed for optimal synthesis of proteins described herein.It would be apparent to one of ordinary skill in the art whichadditional elements to use.

Factors of importance in selecting a particular plasmid or viral vectorinclude, but are not limited to, the ease with which recipient cellsthat contain the vector are recognized and selected from those recipientcells which do not contain the vector; the number of copies of thevector which are desired in a particular host; and whether it isdesirable to be able to “shuttle” the vector between host cells ofdifferent species.

Once the vector(s) is constructed to include a DNA sequence forexpression, it may be introduced into an appropriate host cell by one ormore of a variety of suitable methods that are known in the art,including but not limited to, for example, transformation, transfection,conjugation, protoplast fusion, electroporation, calciumphosphate-precipitation, direct microinjection, etc.

Host cells may either be prokaryotic or eukaryotic. Examples ofeukaryotic host cells include, for example, mammalian cells, such ashuman, monkey, mouse, and Chinese hamster ovary (CHO) cells. Such cellsfacilitate post-translational modifications of proteins, including, forexample, correct folding or glycosylation. Additionally, yeast cells canalso be used to express fusion polypeptides of the invention. Like mostmammalian cells, yeast cells also enable post-translationalmodifications of proteins, including, for example, glycosylation. Anumber of recombinant DNA strategies exist which utilize strong promotersequences and high copy number plasmids that can be utilized forproduction of proteins in yeast. Yeast transcription and translationmachinery can recognize leader sequences on cloned mammalian geneproducts, thereby enabling the secretion of peptides bearing leadersequences (i.e., pre-peptides). A particularly preferred method ofhigh-yield production of the fusion polypeptides of the invention isthrough the use of dihydrofolate reductase (DHFR) amplification inDHFR-deficient CHO cells, by the use of successively increasing levelsof methotrexate as described in U.S. Pat. No. 4,889,803. The polypeptideobtained may be in a glycosylated form.

After the introduction of one or more vector(s), host cells are usuallygrown in a selective medium, which selects for the growth ofvector-containing cells. Purification of the recombinant proteins can becarried out by any of the methods known in the art or described herein,for example, any conventional procedures involving extraction,precipitation, chromatography and electrophoresis. A furtherpurification procedure that may be used for purifying proteins isaffinity chromatography using monoclonal antibodies which bind a targetprotein. Generally, crude preparations containing a recombinant proteinare passed through a column on which a suitable monoclonal antibody isimmobilized. The protein usually binds to the column via the specificantibody while the impurities pass through. After washing the column,the protein is eluted from the gel by changing pH or ionic strength, forexample.

4.4. Assays for Assessing Fusion Polypeptide Activity

Assays described herein (See Example 2) and those known in the art canbe used for detecting Klotho or FGF activity of the fusion polypeptidesof the invention. Suitable activity assays include receptor bindingassays, cellular proliferation assays and cell signaling assays. Forexample, a binding assay which may be used for determining whether afusion polypeptide has Klotho or FGF activity includes, assaying thebinding of a fusion polypeptide to an FGF receptor. FGF receptor bindingassays include, but are not limited to, both competitive andnon-competitive assay. For example, FGF receptor binding can be detectedby contacting cells expressing an FGF receptor with a labeled FGF (forexample, radio-active label) and increasing concentrations of anunlabeled Klotho-FGF fusion polypeptide. The two ligands that competefor binding to the same receptor are added to a reaction mixturecontaining the cell. The cells are subsequently washed and labeled FGFis measured. A decrease in the amount of the labeled FGF to its receptorin the presence of the unlabeled fusion polypeptide is indicative ofbinding of the Klotho-FGF fusion polypeptide to the receptor.Alternatively, the Klotho-FGF fusion polypeptide may be labeled anddirect binding of the fusion polypeptide to the cell is detected.

Klotho or FGF activity can also be measured by determining whether thefusion polypeptide induces a cellular response. For example, in someembodiments, an assay for detecting the biological activity of aKlotho-FGF fusion polypeptide involves contacting cells which express anFGF receptor with a fusion polypeptide, assaying a cellular responsesuch as, for example, cell proliferation or Egr-1 activation, myotubediameter in C2C12 cells, and comparing the cellular response in thepresence and absence of the fusion polypeptide. An increase in thecellular response in the presence of the fusion polypeptide complexrelative to the absence indicates that the fusion polypeptide hasbiological activity. Also, an increase in a downstream signaling eventfrom the receptor can also be measured as indicia of biological activity(e.g., phosphorylation of FGFR, FRS2, ERK1/2, p70S6K etc.).

4.5 Pharmaceutical Compositions and Methods of Treatment

The invention also pertains to pharmaceutical compositions containingone or more fusion polypeptides of the invention and a pharmaceuticallyacceptable diluent or carrier. The pharmaceutical compositions canfurther include a pharmaceutically effective dose of heparin. Suchpharmaceutical compositions may be included in a kit or container. Suchkit or container may be packaged with instructions pertaining to theextended in vivo half-life or the in vitro shelf life of the fusionpolypeptides. Optionally associated with such kit or container(s) can bea notice in the form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of manufacture, use or salefor human administration. Such compositions may be used in methods oftreating, preventing, or ameliorating a disease or a disease symptom(e.g., age-related condition or metabolic disorder) in a patient,preferably a mammal and most preferably a human, by administering thepharmaceutical composition to the patient.

In general, a therapeutically effective amount of a pharmaceuticalcomposition of the invention is from about 0.0001 mg/kg to 0.001 mg/kg;0.001 mg/kg to about 10 mg/kg body weight or from about 0.02 mg/kg toabout 5 mg/kg body weight. Commonly, a therapeutically effective amountof a fusion polypeptide is from about 0.001 mg to about 0.01 mg, about0.01 mg to about 100 mg, or from about 100 mg to about 1000 mg, forexample. Preferably, a therapeutically effective amount of a fusionpolypeptide is from about 0.001 mg/kg to 2 mg/kg.

The optimal pharmaceutical formulations for a fusion polypeptide can bedetermined by one or ordinary skilled in the art depending upon theroute of administration and desired dosage. (See, for example,Remington's Pharmaceutical Sciences, 18th Ed. (1990), Mack PublishingCo., Easton, Pa., the entire disclosure of which is hereby incorporatedby reference).

The fusion polypeptides of the invention may be administered as apharmaceutical composition that may be in the form of a solid, liquid orgas (aerosol). Typical routes of administration may include, withoutlimitation, oral, topical, parenteral, sublingual, rectal, vaginal,intradermal and intranasal. Parenteral administration includessubcutaneous injections, intravenous, intramuscular, intraperitoneal,intrapleural, intrasternal injection or infusion techniques. Preferably,the compositions are administered parenterally. More preferably, thecompositions are administered intravenously. Pharmaceutical compositionsof the invention can be formulated so as to allow a polypeptide of theinvention to be bioavailable upon administration of the composition to asubject. Compositions can take the form of one or more dosage units,where, for example, a tablet can be a single dosage unit, and acontainer of a polypeptide of the invention in aerosol form can hold aplurality of dosage units.

Materials used in preparing the pharmaceutical compositions can benon-toxic in the amounts used. It will be evident to those of ordinaryskill in the art that the optimal dosage of the active ingredient(s) inthe pharmaceutical composition will depend on a variety of factors.Relevant factors include, without limitation, the type of subject (e.g.,human), the overall health of the subject, the type of age-relatedcondition or metabolic disorder the subject in need of treatment of, theuse of the composition as part of a multi-drug regimen, the particularform of the polypeptide of the invention, the manner of administration,and the composition employed.

The pharmaceutically acceptable carrier or vehicle may be particulate,so that the compositions are, for example, in tablet or powder form. Thecarrier(s) can be liquid, with the compositions being, for example, anoral syrup or injectable liquid. In addition, the carrier(s) can begaseous, so as to provide an aerosol composition useful in, e.g.,inhalatory administration.

The term “carrier” refers to a diluent, adjuvant or excipient, withwhich a polypeptide of the invention is administered. Suchpharmaceutical carriers can be liquids, such as water and oils,including those of petroleum, animal, vegetable or synthetic origin,such as peanut oil, soybean oil, mineral oil, sesame oil and the like.The carriers can be saline, gum acacia, gelatin, starch paste, talc,keratin, colloidal silica, urea, and the like. In addition, auxiliary,stabilizing, thickening, lubricating and coloring agents can be used. Inone embodiment, when administered to a subject, the polypeptides of theinvention and pharmaceutically acceptable carriers are sterile. Water isa preferred carrier when the polypeptide of the invention isadministered intravenously. Saline solutions and aqueous dextrose andglycerol solutions can also be employed as liquid carriers, particularlyfor injectable solutions. Suitable pharmaceutical carriers also includeexcipients such as starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The present compositions, if desired, canalso contain minor amounts of wetting or emulsifying agents, or pHbuffering agents.

The composition may be intended for oral administration, and if so, thecomposition is preferably in solid or liquid form, where semi-solid,semi-liquid, suspension and gel forms are included within the formsconsidered herein as either solid or liquid.

As a solid composition for oral administration, the composition can beformulated into a powder, granule, compressed tablet, pill, capsule,chewing gum, wafer or the like form. Such a solid composition typicallycontains one or more inert diluents. In addition, one or more of thefollowing can be present: binders such as ethyl cellulose,carboxymethylcellulose, microcrystalline cellulose, or gelatin;excipients such as starch, lactose or dextrins, disintegrating agentssuch as alginic acid, sodium alginate, Primogel, corn starch and thelike; lubricants such as magnesium stearate or Sterotex; glidants suchas colloidal silicon dioxide; sweetening agents such as sucrose orsaccharin, a flavoring agent such as peppermint, methyl salicylate ororange flavoring, and a coloring agent.

When the pharmaceutical composition is in the form of a capsule, e.g., agelatin capsule, it can contain, in addition to materials of the abovetype, a liquid carrier such as polyethylene glycol, cyclodextrin or afatty oil.

The pharmaceutical composition can be in the form of a liquid, e.g., anelixir, syrup, solution, emulsion or suspension. The liquid can beuseful for oral administration or for delivery by injection. Whenintended for oral administration, a composition can contain one or moreof a sweetening agent, preservatives, dye/colorant and flavour enhancer.In a composition for administration by injection, one or more of asurfactant, preservative, wetting agent, dispersing agent, suspendingagent, buffer, stabilizer and isotonic agent can also be included.

The liquid compositions of the invention, whether they are solutions,suspensions or other like form, can also include one or more of thefollowing: sterile diluents such as water for injection, salinesolution, preferably physiological saline, Ringer's solution, isotonicsodium chloride, fixed oils such as synthetic mono or digylcerides whichcan serve as the solvent or suspending medium, polyethylene glycols,glycerin, cyclodextrin, propylene glycol or other solvents;antibacterial agents such as benzyl alcohol or methyl paraben;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. A parenteral composition can be enclosed inan ampoule, a disposable syringe or a multiple-dose vial made of glass,plastic or other material. Physiological saline is a preferred adjuvant.An injectable composition is preferably sterile.

The pharmaceutical compositions contain an effective amount of acompound of the invention (e.g., fusion polypeptide) such that asuitable dosage will be obtained. The pharmaceutical compositions maycontain the known effective amount of the compounds as currentlyprescribed for their respective disorders.

The route of administration of the polypeptide of the invention used inthe prophylactic and/or therapeutic regimens which will be effective inthe prevention, treatment, and/or management of a age-related conditionor metabolic disorder can be based on the currently prescribed routes ofadministration for other therapeutics known in the art. The polypeptidesof the invention can be administered by any convenient route, forexample, by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.). Administration can be systemic or local.Various delivery systems are known, e.g., microparticles, microcapsules,capsules, etc., and may be useful for administering a polypeptide of theinvention. More than one polypeptides of the invention may beadministered to a subject. Methods of administration may include, butare not limited to, oral administration and parenteral administration;parenteral administration including, but not limited to, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, sublingual, intranasal, intracerebral, intraventricular,intrathecal, intravaginal, transdermal, rectally, by inhalation, ortopically to the ears, nose, eyes, or skin.

The polypeptides of the invention may be administered parenterally.Specifically, the polypeptides of the invention may be administeredintravenously.

Pulmonary administration can also be employed, e.g., by use of aninhaler or nebulizer, and formulation with an aerosolizing agent, or viaperfusion in a fluorocarbon or synthetic pulmonary surfactant. Thepolypeptides of the invention can also be formulated as a suppository,with traditional binders and carriers such as triglycerides.

The polypeptides of the invention can be delivered in a controlledrelease system. For example, a pump can be used (see Sefton, CRC Crit.Ref. Biomed. Eng. 1987, 14, 201; Buchwald et al., Surgery 1980, 88: 507;Saudek et al., N. Engl. J. Med. 1989, 321: 574). Polymeric materials canalso be used for controlled release of the polypeptides of the invention(see Medical Applications of Controlled Release, Langer and Wise (eds.),CRC Pres., Boca Raton, Fla., 1974; Controlled Drug Bioavailability, DrugProduct Design and Performance, Smolen and Ball (eds.), Wiley, New York,1984; Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 1983,23, 61; see also Levy et al., Science 1985, 228, 190; During et al.,Ann. Neurol., 1989, 25, 3511; Howard et al., J. Neurosurg., 1989, 71,105). Specifically, a controlled-release system can be placed inproximity of the target of the polypeptides of the invention, e.g., thebrain, thus requiring only a fraction of the systemic dose (see, e.g.,Goodson, in Medical Applications of Controlled Release, supra, vol. 2,1984, pp. 115-138). Other controlled-release systems discussed in thereview by Langer (Science 1990, 249, 1527-1533) can be used.

Polymeric materials used to achieve controlled or sustained release ofthe polypeptides of the invention are disclosed, e.g., in U.S. Pat. No.5,679,377; U.S. Pat. No. 5,916,597; U.S. Pat. No. 5,912,015; U.S. Pat.No. 5,989,463; U.S. Pat. No. 5,128,326; PCT Publication No. WO 99/15154;and PCT Publication No. WO 99/20253. Examples of polymers used insustained release formulations include, but are not limited to,poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate),poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylicacid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone),poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides(PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters.Preferably, the polymer used in a sustained release formulation isinert, free of leachable impurities, stable on storage, sterile, andbiodegradable.

In general, a therapeutically effective amount of a pharmaceuticalcomposition of the invention is from about 0.0001 mg/kg to 0.001 mg/kg;0.001 mg/kg to about 10 mg/kg body weight or from about 0.02 mg/kg toabout 5 mg/kg body weight.

In other embodiments, the prophylactic and/or therapeutic regimeninvolves administering to a patient one or more doses of an effectiveamount of a polypeptide of the invention, wherein the dose of aneffective amount achieves a plasma level of at least 0.01 μg mL to atleast 400 μg/mL of the polypeptide of the invention.

A prophylactic and/or therapeutic regimen may involve administering to apatient a plurality of doses of an effective amount of a polypeptide ofthe invention, wherein the plurality of doses maintains a plasma levelof at least 0.01 μg/mL, to 400 μg/mL of the polypeptide of theinvention. The prophylactic and/or therapeutic regimen may beadministered for at least 1 day, 1 month, 2 months, 3 months, 4 months,5 months, 6 months, 7 months, 8 months or 9 months.

The prophylactic and/or therapeutic regimen may involve administrationof a polypeptide of the invention in combination with one or moreadditional therapeutics. The recommended dosages of the one or moretherapeutics currently used for the prevention, treatment, and/ormanagement of an age-related condition or metabolic disorder can beobtained from any reference in the art including, but not limited to,Hardman et al., eds., Goodman & Gilman's The Pharmacological Basis OfBasis Of Therapeutics, 10th ed., Mc-Graw-Hill, New York, 2001;Physician's Desk Reference (60^(th) ed., 2006), which is incorporatedherein by reference in its entirety.

The invention includes methods of treating disorders wherein agonisticactivity of Klotho protein and FGF are desirable. Examples of suchmethods of the invention include, but are not limited to age-relatedcondition or metabolic disorders.

The invention includes methods for treating or preventing an age-relatedcondition in an individual. An individual in need of treatment isadministered a pharmacologically effective dose of a pharmaceuticalcomposition containing a Klotho fusion polypeptide, having at least oneextracellular subdomain of a Klotho protein and a fibroblast growthfactor so as to treat or prevent the age-related condition. In someembodiments, the Klotho fusion polypeptide is coadministered with apharmacologically effective dose of heparin. Age-related conditionsinclude sarcopenia, skin atrophy, muscle wasting, brain atrophy,atherosclerosis, arteriosclerosis, pulmonary emphysema, osteoporosis,osteoarthritis, immunologic incompetence, high blood pressure, dementia,Huntington's disease, Alzheimer's disease, cataracts, age-relatedmacular degeneration, prostate cancer, stroke, diminished lifeexpectancy, memory loss, wrinkles, impaired kidney function, andage-related hearing loss. In some embodiments, the Klotho fusionpolypeptide contains at least one extracellular domain of an alphaKlotho protein. In a particular embodiment, a Klotho fusion proteincontaining at least one extracellular domain of alpha Klotho protein andfibroblast growth factor 23 is administered to an individual in need oftreatment for muscle wasting.

The invention is also directed to a method for treating or preventing ametabolic disorder in an individual. An individual in need of treatmentis administered a pharmacologically effective dose of a pharmaceuticalcomposition containing a Klotho fusion polypeptide, having at least oneextracellular subdomain of a Klotho protein and a fibroblast growthfactor so as to treat the metabolic disorder. In some embodiments, theKlotho fusion polypeptide is coadministered with a pharmacologicallyeffective dose of heparin. The method may be used in the treatment orprevention of Type II Diabetes, Metabolic Syndrome, hyperglycemia, andobesity. In a particular embodiment, a Klotho fusion protein containingat least one extracellular domain of a beta-Klotho protein andfibroblast growth factor 21 is administered to an individual in need oftreatment for a metabolic disorder.

The invention also provides methods for treating or preventinghyperphosphatemia or calcinosis in an individual. An individual in needof treatment is administered a pharmacologically effective dose of apharmaceutical composition containing a Klotho fusion polypeptide,having at least one extracellular subdomain of a Klotho protein and afibroblast growth factor so as to treat hyperphosphatemia or calcinosis.In some embodiments, the Klotho fusion polypeptide is coadministeredwith a pharmacologically effective dose of heparin. In a particularembodiment, a Klotho fusion protein containing at least oneextracellular domain of an alpha Klotho protein and fibroblast growthfactor 23 is administered to an individual in need of treatment for ahyperphosphatemia or calcinosis.

The invention is also directed to a method for treating or preventingchronic renal disease or chronic renal failure in an individual. Anindividual in need of treatment is administered a pharmacologicallyeffective dose of a pharmaceutical composition containing a Klothofusion polypeptide, having at least one extracellular subdomain of aKlotho protein and a fibroblast growth factor so as to treat chronicrenal disease or chronic renal failure. In some embodiments, the Klothofusion polypeptide is coadministered with a pharmacologically effectivedose of heparin. In some embodiments, a Klotho fusion protein containingat least one extracellular domain of an alpha Klotho protein isadministered to an individual in need of treatment for chronic renaldisease or chronic renal failure.

The invention also includes methods for treating or preventing cancer inan individual. An individual in need of treatment is administered apharmacologically effective dose of a pharmaceutical compositioncontaining a Klotho fusion polypeptide, having at least oneextracellular subdomain of a Klotho protein and a fibroblast growthfactor so as to treat cancer. The method may be used in the treatment orprevention of breast cancer. In some embodiments, the Klotho fusionpolypeptide is coadministered with a pharmacologically effective dose ofheparin. In some embodiments, a Klotho fusion protein containing atleast one extracellular domain of an alpha Klotho protein isadministered to an individual in need of treatment for cancer.

In methods of treating disorders by administering a pharmaceuticalcomposition containing a Klotho fusion polypeptide, the Klotho fusionpolypeptide has at least one extracellular subdomain of a Klotho proteinand a fibroblast growth factor. In a particular embodiment, the Klothofusion protein contains at least one extracellular domain of a betaKlotho protein and fibroblast growth factor 21.

The Klotho fusion polypeptide composition can be administered accordingto any method of administration known to those of skill in the art anddescribed herein. Preferred methods of administration includesubcutaneous or intravenous. Other effective modes of administration aredescribed herein.

4.6. Methods of Treatment and Assays for Assessing Efficacy

Methods of the invention which provide administering the Klotho fusionpolypeptide to an individual can be used to treat a variety of disordersincluding an age-related disorder or a metabolic disorder. Without beinglimited by any particular theory, Klotho/FGF fusion polypeptides may beused to treat disorders in which there is dysregulation of Klotho orFGF. Exemplary disorders include metabolic disorders and age-relateddisorders. For example, both FGF23 or Klotho knock-out mice display avariety of similar phenotypes including, low physical activity, growthretardation, muscle wasting, skin atrophy, atherosclerosis, short lifespans, etc. (See Razzaque and Lanske, J. of Endrocrinology, 194:1-10(2007), which is herein incorporated by reference).

In particular, Klotho/FGF23 fusion polypeptides of the invention areparticularly useful in the treatment of aging-related disorders,including muscle wasting. Without being bound to theory, the ability ofKlotho and FGF23 to control mineral (e.g., phosphate and calcium) andvitamin D homeostasis may be the means by which these proteins modulateaging and muscle atrophy.

On the other hand, Klotho/FGF19 fusion polypeptides and Klotho/FGF21fusion polypeptides of the invention may be used for treating ametabolic disorder. For example, beta-Klotho and FGF19 have been shownto control bile acid homeostasis by regulating cholesterol7-α-hydroxylase (CYP7A1). A non-limiting example of bile homeostasisdisorder is cholestasis. The beta-Klotho and FGF21 have been shown toinduce lipolysis in adipocytes and, therefore, reduced fat storage andincreased glucose uptake. Non-limiting examples of lipolysis/fat storagedisorders are obesity and associated metabolic and carciovasculardiseases.

Based at least in part on the finding that FGF23 is able to stimulateexcretion of phosphate in the urine and thereby reduce phosphate levelsin the serum, Klotho-FGF23 fusion polypeptides of the invention can beused for treating or preventing hyperphosphatemia or calcinosis in anindividual. For example, it has been shown that a homozygous missensemutation in Klotho resulting in a deficiency in Klotho in a patient cancause severe tumoral calcinosis and artery calcification (Ichikawa etal., J. Clin. Invest. 117:2684-2691 (2007), which is herein incorporatedby reference). An individual is administered a pharmacologicallyeffective dose of a pharmaceutical composition containing the Klothofusion polypeptide, having at least one extracellular subdomain of aKlotho protein and a fibroblast growth factor so as to treat or preventhyperphosphatemia or calcinosis. In particular, a Klotho fusionpolypeptide containing at least one extracellular domain of an alphaKlotho protein and a fibroblast growth factor is useful for treatinghyperphosphatemia or calcinosis.

Klotho fusion polypeptides of the invention can also be used fortreating or preventing chronic renal disease or chronic renal failure inan individual. For example, it has been shown that Klotho expression isreduced in kidney of patients with chronic renal failure, compared tothat in unaffected kidneys (Koh et al., Biochem. Biophys. Res. Comm.280:1015-1020 (2001), which is herein incorporated by reference). Anindividual is administered a pharmacologically effective dose of apharmaceutical composition containing the Klotho fusion polypeptide,having at least one extracellular subdomain of a Klotho protein and afibroblast growth factor so as to treat or prevent chronic renal diseaseor chronic renal failure. In particular, a Klotho fusion polypeptidecontaining at least one extracellular domain of an alpha Klotho proteinis useful for treating chronic renal disease or chronic renal failure.

Klotho fusion polypeptides of the invention can also be used fortreating or preventing cancer in an individual. For example, it has beenshown that Klotho expression is reduced in breast cancer tissue,compared to normal breast cancer tissue (Wolf et al., Oncogene (2008)advance online publication, which is herein incorporated by reference).An individual is administered a pharmacologically effective dose of apharmaceutical composition containing the Klotho fusion polypeptide,having at least one extracellular subdomain of a Klotho protein and afibroblast growth factor so as to treat or prevent cancer or breastcancer. In particular, a Klotho fusion protein containing at least oneextracellular domain of an alpha Klotho protein is useful for treatingcancer or breast cancer.

Methods for evaluating the efficacy and/or determining the effectivedose of a Klotho fusion polypeptide of the invention on an age-relateddisorder or metabolic disorder include organismal based assays, e.g.,using a mammal (e.g., a mouse, rat, primate, or some other non-human),or other animal (e.g., Xenopus, zebrafish, or an invertebrate such as afly or nematode). The Klotho fusion polypeptide can be administered tothe organism once or as a regimen (regular or irregular). A parameter ofthe organism is then evaluated, e.g., an age-associated parameter.Klotho fusion polypeptides that are of interest result in a change inthe parameter relative to a reference, e.g., a parameter of a controlorganism. Other parameters (e.g., related to toxicity, clearance, andpharmacokinetics) can also be evaluated.

The Klotho fusion polypeptide of the invention may be evaluated using ananimal that has a particular disorder, e.g., a disorder describedherein, e.g., an age-related disorder, a metabolic disorder. Thesedisorders can also provide a sensitized system in which the testpolypeptide's effects on physiology can be observed. Exemplary disordersinclude: denervation, disuse atrophy; metabolic disorders (e.g.,disorder of obese and/or diabetic animals such as db/db mouse and ob/obmouse); cerebral, liver ischemia; cisplatin/taxol/vincristine models;various tissue (xenograph) transplants; transgenic bone models; painsyndromes (include inflammatory and neuropathic disorders); Paraquat,genotoxic, and oxidative stress models; and tumor I models.

For measuring an age-related disorder, the animal model can be an animalthat has an altered phenotype when calorically restricted. For example,F344 rats provide a useful assay system for evaluating a Klotho fusionpolypeptide. When calorically restricted, F344 rats have a 0 to 10%incidence of nephropathy. However, when fed ad libitum, they have a 60to 100% incidence of nephropathy.

To evaluate a Klotho fusion polypeptide of the invention, it isadministered to the animal (e.g., an F344 rat or other suitable animal)and a parameter of the animal is evaluated, e.g., after a period oftime. The animal can be fed ad libitum or normally (e.g., not undercaloric restriction, although some parameters can be evaluated undersuch conditions). Typically, a cohort of such animals is used for theassay. Generally, a test polypeptide can be indicated as favorablyaltering lifespan regulation in the animal if the test polypeptideaffects the parameter in the direction of the phenotype of a similaranimal subject to caloric restriction. Such test polypeptides may causeat least some of the lifespan regulatory effects of caloric restriction,e.g., a subset of such effects, without having to deprive the organismof caloric intake.

The parameter to be tested may be an age-associated or diseaseassociated parameter, e.g., a symptom of the disorder associated withthe animal model. For example, the test polypeptide can be administeredto a SH Rat, and blood pressure is monitored. A test polypeptide that isfavorably indicated can cause an amelioration of the symptom relative toa similar reference animal not treated with the polypeptide. Otherparameters relevant to a disorder or to aging can include: antioxidantlevels (e.g. antioxidant enzyme levels or activity), stress resistance(e.g., paraquat resistance), core body temperature, glucose levels,insulin levels, thyroid-stimulating hormone levels, prolactin levels,and leutinizing hormone levels.

To measure the effectiveness of the polypeptides of the invention fortreating an age-related disorder, an animal having decreased Klothoexpression may be used, e.g., mouse with a mutant Klotho; See Kuroo, etal. Nature, 390; 45 (1997) and U.S. Pub. No. 2003/0119910, both of whichare herein incorporated by reference in their entirety. For example, thetest polypeptide is administered to the mutant mouse and age-relatedparameters are monitored. A test polypeptide that is favorably indicatedcan cause an amelioration of the symptom relative to a similar referenceanimal not treated with the polypeptide. A parameter relevant to ametabolic disorder or to aging can be assessed by measurement of bodyweight, examination on the acquisition of reproductive ability,measurement of blood sugar level, observation of life span, observationof skin, observation of motor functions such as walking, and the like.The assessment can also be made by measurement of thymus weight,observation of the size of calcified nodules formed on the inner surfaceof thoracic cavity, and the like. Further, quantitative determination ofmRNA for the Klotho gene or Klotho protein is also useful for theassessment.

Still other in vivo models and organismal assays include evaluating ananimal for a metabolic parameter, e.g., a parameter relevant to aninsulin disorder, type II diabetes. Exemplary metabolic parametersinclude: glucose concentration, insulin concentration, and insulinsensitivity.

Another exemplary system features tumors, e.g., in an animal model. Thetumors can be spontaneous or induced. For example, the tumors can bedeveloped from cells that have a variety of genetic constitutions, e.g.,they can be p53+ or p53−. It is also possible to use organisms that anautoimmune disorder, e.g., an NZB mouse, which is predisposed to SLE. Toevaluate features of bone disease, it is possible, for example, to usean animal that has an ovariectomy as a model, e.g., for osteoporosis.Similarly, for joint disease, the model can be based on adjuvantarthritis (e.g., mice can be immunized with cartilage proteoglycans,high mobility group proteins, streptococcal cell wall material, orcollagens); for kidney disease, kd/kd mice can be used. Animal models ofcognition, particularly learning and memory are also available. Animalmodels of diabetes and its complications are also available, e.g., thestreptozotocin model. Canine models can be used, for example, forevaluating stroke and ischemia.

In assessing whether a test polypeptide is capable of altering life spanregulation, a number of age-associated parameters or biomarkers can bemonitored or evaluated. Exemplary age associated parameters include: (i)lifespan of the cell or the organism; (ii) presence or abundance of agene transcript or gene product in the cell or organism that has abiological age dependent expression pattern; (iii) resistance of thecell or organism to stress; (iv) one so or more metabolic parameters ofthe cell or organism (exemplary parameters include circulating insulinlevels, blood glucose levels; fat content; core body temperature and soforth); (v) proliferative capacity of the cell or a set of cells presentin the organism; and (vi) physical appearance or behavior of the cell ororganism.

The term “average lifespan” refers to the average of the age of death ofa cohort of organisms. In some cases, the “average lifespan” is assessedusing a cohort of genetically identical organisms under controlledenvironmental conditions. Deaths due to mishap are discarded. Whereaverage lifespan cannot be determined (e.g., for humans) undercontrolled environmental conditions, reliable statistical information(e.g., from actuarial tables) for a sufficiently large population can beused as the average lifespan.

Characterization of molecular differences between two such organisms,e.g., one reference organism and one organism treated with a Klothofusion polypeptide can reveal a difference in the physiological state ofthe organisms. The reference organism and the treated organism aretypically the same chronological age. The term “chronological age” asused herein refers to time elapsed since a preselected event, such asconception, a defined embryological or fetal stage, or, more preferably,birth. A variety of criteria can be used to determine whether organismsare of the “same” chronological age for the comparative analysis.Typically, the degree of accuracy required is a function of the averagelifespan of a wildtype organism. For example, for the nematode C.elegans, for which the laboratory wildtype strain N2 lives an to averageof about 16 days under some controlled conditions, organisms of the sameage may have lived for the same number of days. For mice, organism ofthe same age may have lived for the same number of weeks or months; forprimates or humans, the same number of years (or within 2, 3, or 5years); and so forth. Generally, organisms of the same chronological agemay have lived for an amount of time within 15, 10, 5, 3, 2 or 1% of theaverage lifespan of a wildtype organism of that species. Preferably, theorganisms are adult organisms, e.g., the organisms have lived for atleast an amount of time in which the average wildtype organism hasmatured to an age at which it is competent to reproduce.

The organismal screening assay can be performed before the organismsexhibit overt physical features of aging. For example, the organisms maybe adults that have lived only 10, 30, 40, 50, 60, or 70% of the averagelifespan of a wildtype organism of the same species. Age-associatedchanges in metabolism, immune competence, and chromosomal structure havebeen reported. Any of these changes can be evaluated, either in a testsubject (e.g., for an organism based assay), or for a patient (e.g.,prior, during or after treatment with a therapeutic described herein.

A marker associated with caloric restriction can also be evaluated in asubject organism of a screening assay (or a treated subject). Althoughthese markers may not be age-associated, they may be indicative of aphysiological state that is altered when the Klotho pathway ismodulated. The marker can be an mRNA or protein whose abundance changesin calorically restricted animals. WO01/12851 and U.S. Pat. No.6,406,853 describe exemplary markers. Cellular models derived from cellsof an animal described herein or analogous to an animal model describedherein can be used for a cell-based assay.

Models for evaluating the effect of a test polypeptide on muscle atrophyinclude: 1) rat medial gastrocnemius muscle mass loss resulting fromdenervation, e.g., by severing the right sciatic nerve at mid-thigh; 2)rat medial gastrocnemius muscle mass loss resulting from immobilization,e.g., by fixed the right ankle joint at 90 degrees of flexion; 3) ratmedial gastrocnemius muscle mass loss resulting from hind limbsuspension; (see, e.g., U.S. 2003-0129686); 4) skeletal muscle atrophyresulting from treatment with the cachectic cytokine, interleukin-1(IL-1) (R. N. Cooney, S. R. Kimball, T. C. Vary, Shock 7, 1-16 (1997));and 5) skeletal muscle atrophy resulting from treatment with theglucocorticoid, dexamethasone (A. L. Goldberg, J Biol Chem 244, 3223-9(1969).)

Exemplary animal models for AMD include: laser-induced mouse modelsimulating exudative (wet) macular degeneration Bora et al., Proc. Natl.Acad. Sci. USA., 100:2679-84 (2003); a transgenic mouse expressing amutated form of cathepsin D resulting in features associated with the“geographic atrophy” form of AMD (Rakoczy et al., Am. J. Pathol.,161:1515-24 (2002)); and a transgenic mouse over expressing VEGF in theretinal pigment epithelium resulting in CNV. Schwesinger et al., Am. J.Pathol. 158:1161-72 (2001).

Exemplary animal models of Parkinson's disease include primates renderedParkinsonian by treatment with the dopaminergic neurotoxin 1-methyl-4phenyl 1,2,3,6-tetrahydropyridine (MPTP) (see, e.g., U.S. PatentPublication No. 20030055231 and Wichmann et al., Ann. N.Y. Acad. Sci.,991:199-213 (2003); 6-hydroxydopamine-lesioned rats (e.g., Lab. Anim.Sci., 49:363-71 (1999)); and transgenic invertebrate models (e.g., Laksoet al., J. Neurochem. 86:165-72 (2003) and Link, Mech. Ageing Dev.,122:1639-49 (2001)).

Exemplary molecular models of Type II diabetes include: a transgenicmouse having defective Nkx-2.2 or Nkx-6.1; (U.S. Pat. No. 6,127,598);Zucker Diabetic Fatty fa/fa (ZDF) rat. (U.S. Pat. No. 6,569,832); andRhesus monkeys, which spontaneously develop obesity and subsequentlyfrequently progress to overt type 2 diabetes (Hotta et al., Diabetes,50:1126-33 (2001); and a transgenic mouse with a dominant-negative IGF-Ireceptor (KR-IGF-IR) having Type 2 diabetes-like insulin resistance.

Exemplary animal and cellular models for neuropathy include: vincristineinduced sensory-motor neuropathy in mice (U.S. Pat. No. 5,420,112) orrabbits (Ogawa et al., Neurotoxicology, 21:501-11 (2000)); astreptozotocin (STZ)-diabetic rat for study of autonomic neuropathy(Schmidt et al., Am. J. Pathol., 163:21-8 (2003)); and a progressivemotor neuropathy (pmn) mouse (Martin et al., Genomics, 75:9-16 (2001)).

Exemplary animal models of hyperphosphatemia or tumoral calcinosisinclude Klotho knockout mice and FGF23 knockout mice (Yoshida et al.,Endocrinology 143:683-689 (2002)).

Exemplary animal models of chronic renal disease or chronic renalfailure include COL4A3+/−mice (Beirowski et al., J. Am. Soc. Nephrol.17:1986-1994 (2006)).

Exemplary animal models of cancer include the transplantation orimplantation of cancer cells or tissue into nude mice, as is known inthe art (Giovanella et al., Adv. Cancer Res. 44:69-120 (1985)). Forexample, animal models of breast cancer include nude mice transplantedor implanted with breast cancer cells or tissue (e.g., Yue et al.,Cancer Res. 54:5092-5095 (1994); Glinsky et al., Cancer Res.56:5319-5324 (1996); Visonneau Am. J. Path. 152:1299-1311 (1998)).

The compositions can be administered to a subject, e.g., an adultsubject, particularly a healthy adult subject or a subject having anage-related disease. In the latter case, the method can includeevaluating a subject, e.g., to characterize a symptom of an age-relateddisease or other disease marker, and thereby identifying a subject ashaving a neurodegenerative disease, e.g., Alzheimer's or an age-relateddisease or being pre-disposed to such a disease.

Skeletal Muscle Atrophy

Methods of the invention which provide administering the Klotho fusionpolypeptide to an individual can be used to treat skeletal muscleatrophy. Muscle atrophy includes numerous neuromuscular, metabolic,immunological and neurological disorders and diseases as well asstarvation, nutritional deficiency, metabolic stress, diabetes, aging,muscular dystrophy, or myopathy. Muscle atrophy occurs during the agingprocess. Muscle atrophy also results from reduced use or disuse of themuscle. Symptoms include a decline in skeletal muscle tissue mass. Inhuman males, muscle mass declines by one-third between the ages of 50and 80. Some molecular features of muscle atrophy include theupregulation of ubiquitin ligases, and the loss of myofibrillar proteins(Furuno et al., J. Biol. Chem., 265:8550-8557, 1990). The breakdown ofthese proteins can be followed, e.g., by measuring 3-methyl-histidineproduction, which is a specific constituent of actin, and in certainmuscles of myosin (Goodman, Biochem. J. 241:121-12, 1987 and Lowell, etal., Metabolism, 35:1121-112, 1986; Stein and Schluter, Am. J. Physiol.Endocrinol.Metab. 272: E688-E696, 1997). Release of creatine kinase (acell damage marker) (Jackson, et al., Neurology, 41: 101104, 1991) canalso be indicative.

Non-Insulin-Dependent Diabetes

Methods of the invention which provide administering the Klotho fusionpolypeptide to an individual can be used to treat Non-insulin-dependentDiabetes. Non-insulin-dependent Diabetes is also called “adult onset”diabetes and Type 2 diabetes. Type 2 diabetes also includes “non-obesetype 2” and “obese type 2.” Type II diabetes can be characterized by (1)reduced pancreatic-beta-islet-cell secretion of insulin such that lessthan necessary amounts of insulin are produced to keep blood glucoselevels in balance and/or (2) “insulin resistance,” wherein the bodyfails to respond normally to insulin. (U.S. Pat. No. 5,266,561 and U.S.Pat. No. 6,518,069). For example, glucose-stimulated insulin levelstypically fail to rise above 4.0 nmol/L. (U.S. Pat. No. 5,266,561).Exemplary symptoms of Type II diabetes include: hyperglycemia whilefasting (U.S. Pat. No. 5,266,561); fatigue; excessive thirst; frequenturination; blurred vision; and an increased rate of infections.Molecular indications of Type II diabetes include islet amyloiddeposition in the pancreases.

Neuropathy

Neuropathy can include a central and/or peripheral nerve dysfunctioncaused by systemic disease, hereditary condition or toxic agentaffecting motor, sensory, sensorimotor or autonomic nerves. (see, e.g.,US Patent Application No. 20030013771). Symptoms can vary depending uponthe cause of the nerve damage and the particular types of nervesaffected. For example, symptoms of motor neuropathy include clumsinessin performing physical tasks or as muscular weakness, exhaustion afterminor exertion, difficulty in standing or walking and attenuation orabsence of a neuromuscular reflex. (U.S. Patent Application No.20030013771) symptoms of autonomic neuropathy include constipation,cardiac irregularities and attenuation of the postural hypotensivereflex. (U.S. Patent Application No. 20030013771), symptoms of sensoryneuropathy include pain and numbness; tingling in the hands, legs orfeet; and extreme sensitivity to touch, and symptoms of retinopathyinclude blurred vision, sudden loss of vision, black spots, and flashinglights.

Alzheimer's Disease

Methods of the invention which provide administering the Klotho fusionpolypeptide to an individual can be used to treat Alzheimer's Disease(AD). Alzheimer's Disease is a complex neurodegenerative disease thatresults in the irreversible loss of neurons. It provides merely oneexample of a neurodegenerative disease that is also an age-relatedcondition. Clinical hallmarks of Alzheimer's Disease include progressiveimpairment in memory, judgment, orientation to physical surroundings,and language. Neuropathological hallmarks of AD include region-specificneuronal loss, amyloid plaques, and neurofibrillary tangles. Amyloidplaques are extracellular plaques containing the amyloid peptide (alsoknown as Ap, or Ap42), which is a cleavage product of the, 8-amyloidprecursor protein (also known as APP). Neurofibrillary tangles areinsoluble intracellular aggregates composed of filaments of theabnormally hyperphosphorylated microtubule-associated protein, tautAmyloid plaques and neurofibrillary tangles may contribute to secondaryevents that lead to neuronal loss by apoptosis (Clark and Karlawish,Ann. Intern. Med. 138(5):400-410 (2003). For example, p-amyloid inducescaspase-2-dependent apoptosis in cultured neurons (Troy et al. J,Neurosci. 20(4): 1386-1392). The deposition of plaques in viva maytrigger apoptosis of proximal neurons in a similar manner.

A variety of criteria, including genetic, biochemical, physiological,and cognitive criteria, can be used to evaluate AD in a subject.Symptoms and diagnosis of AD are known to medical practitioners. Someexemplary symptoms and markers of AD are presented below. Informationabout these indications and other indications known to be associatedwith AD can be used as an “AD-related parameter.” An AD relatedparameter can include qualitative or quantitative information. Anexample of quantitative information is a numerical value of one or moredimensions, e.g., a concentration of a protein or a tomographic map.Qualitative information can include an assessment, e.g., a physician'scomments or a binary (“yes”/“no”) and so forth. An AD-related parameterincludes information that indicates that the subject is not diagnosedwith AD or does not have a particular indication of AD, e.g., acognitive test result that is not typical of AD or a genetic APOEpolymorphism not associated with AD.

Progressive cognitive impairment is a hallmark of AD. This impairmentcan present as decline in memory, judgment, decision making, orientationto physical surroundings, and language (Nussbaum and Ellis, New Eng J.Med. 348(14):1356 35 1364 (2003)). Exclusion of other forms of dementiacan assist in making a diagnosis of AD. Neuronal death leads toprogressive cerebral atrophy in AD patients. Imaging techniques (e.g.,magnetic resonance imaging, or computer assisted tomography) can be usedto detect AD-associated lesions in the brain and/or brain atrophy.

AD patients may exhibit biochemical abnormalities that result from thepathology of the disease. For example, levels of tan protein in thecerebrospinal fluid is elevated in AD patients (Andreasen, N. et al.Arch Neurol. 58:349-350 (2001)).

Levels of amyloid beta 42 (A,B42) peptide can be reduced in CSF of ADpatients. Levels of Ap42 can be increased in the plasma of AD patients(Ertekein-Taner, N., et al. Science 290:2303 2304 (2000)). Techniques todetect biochemical abnormalities in a sample from a subject includecellular, immunological, and other biological methods known in the art.For general guidance, see, e.g., techniques described in Sambrook &Russell, Molecular Cloning: A Laboratory Manual, 3r Edition, Cold SpringHarbor Laboratory, N.Y. (2001), Ausubel et al., Current Protocols inMolecular Biology (Greene Publishing Associates and Wiley Interscience,N.Y. (1989), (Harrow, E. and Lane, D. (1988) Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.),and updated editions thereof.

For example, antibodies, other immunoglobulins, and other specificbinding ligands can be used to detect a biomolecule, e.g., a protein orother antigen associated with AD. For example, one or more specificantibodies can be used to probe a sample. Various formats are possible,e.g., ELISAs, fluorescence-based assays, Western blots, and proteinarrays. Methods of producing polypeptide arrays are described in theart, e.g., in De Wildt et al. (2000). Nature Biotech. 18, 989-994;Lucking et al. (1999). Anal. Biochem. 270, 103-111; Ge, H. (2000).Nucleic Acids Res. 28, e3, I-VII; MacBeath, G., and Schreiber, S. L.(2000). Science 289, 1760 to 1 763; and WO 99/5 1 773A1.

In one assay, a non-human animal model of AD (e.g., a mouse model) isused, e.g., to evaluate a polypeptide or a therapeutic regimen. Forexample, U.S. Pat. No. 6,509,515 describes one such model animal whichis naturally able to be used with learning and memory tests. The animalexpresses an amyloid precursor protein (APP) sequence at a level inbrain tissues such that the animal develops a progressive necrologicdisorder within a short period of time from birth, generally within ayear from birth, preferably within 2 to 6 months, from birth. The APPprotein sequence is introduced into the animal, or an ancestor of theanimal, at an embryonic stage, preferably the one cell, or fertilizedoocyte, stage, and generally not later than about the 8-cell stage. Thezygote or embryo is then developed to term in a pseudo-pregnant asfoster female. The amyloid precursor protein genes are introduced intoan animal embryo so as to be chromosomally incorporated in a state whichresults in super endogenous expression of the amyloid precursor proteinand the development of a progressive necrologic disease in thecortico-limbic areas of the brain, areas of the brain which areprominently affected in progressive necrologic disease states such asAD. The gliosis and clinical manifestations in affected transgenicanimals model necrologic disease. The progressive aspects of theneurologic disease are characterized by diminished exploratory and/orlocomotor behavior and diminished deoxyglucose uptake/utilization andhypertrophic gliosis in the cortico-limbic regions of the brain.Further, the changes that are seen are similar to those that are seen insome aging animals. Other animal models are also described in U.S. Pat.Nos. 5,387,742; 5,877,399; 6,358,752; and 6,187,992.

Parkinson's Disease

Methods of the invention which provide administering the Klotho fusionpolypeptide to an individual can be used to treat Parkinson's Disease.Parkinson's disease includes neurodegeneration of dopaminergic neuronsin the substantia nigra resulting in the degeneration of thenigrostriatal dopamine system that regulates motor function. Thispathology, in turn, leads to motor dysfunctions. (see, e.g., andLotharius et al., Nat. Rev. Neurosci., 3:932-42 (2002)). Exemplary motorsymptoms include: akinesia, stooped posture, gait difficulty, posturalinstability, catalepsy, muscle rigidity, and tremor. Exemplary non-motorsymptoms include: depression, lack of motivation, passivity, dementiaand gastrointestinal dysfunction (see, e.g., Fahn, Ann. N.Y. Acad. Sci.,991:1-14 (2003) and Pfeiffer, Lancet Neurol., 2:107-16 (2003))Parkinson's has been observed in 0.5 to 1 percent of persons 65 to 69years of age and 1 to 3 percent among persons 80 years of age and older.(see, e.g., Nussbaum et al., N. Engl. J. Med., 348:1356-64 (2003)).Molecular markers of Parkinson's disease include reduction in aromatic Lamino acid decarboxylase (AADC) (see, e.g., US App. No. 20020172664);and loss of dopamine content in the nigrostriatal neurons (see, e.g.,Fahn, Ann. N.Y. Acad. Sci., 991:1-14 (2003) and Lotharius et al., Nat.Rev. Neurosci., 3:932-42 (2002)). In some familial cases, PD is linkedto mutations in single genes encoding alpha-synuclein and parkin (an E3ubiquitin ligase) proteins. (e.g., Riess et al., J. Neurol. 250 Suppl1:I3 10 (2003) and Nussbaum et al., N. Engl. J. Med., 348:1356-64(2003)). A missense mutation in a neuron-specific C-terminal ubiquitinhydrolase gene is also associated with Parkinson's. (e.g., Nussbaum etal., N. Engl. J. Med., 348:1356-64 (2003))

Huntington's Disease

Methods of the invention which provide administering the Klotho fusionpolypeptide to an individual can be used to treat Huntington's Disease.Methods for evaluating the efficacy and/or determining the effectivedose of a Klotho fusion polypeptide on Huntington's Disease includeorganismal based assays, e.g., using a mammal (e.g., a mouse, rat,primate, or some other non-human), or other animal (e.g., Xenopus,zebrafish, or an invertebrate such as a fly or nematode). A number ofanimal model system for Huntington's disease are available. See, e.g.,Brouillet, Functional Neurology 15(4): 239-251 (2000); Ona et al. Nature399: 263-267 (1999), Bates et al. Hum Mol Genet. 6(10): 1633-7 (1997);Hansson et al. J. of Neurochemistry 78: 694-703; and Rubinsztein, D. C.,Trends in Genetics, Vol. 1S, No. 4, pp. 202-209 (a review on variousanimal and non-human models of HD).

An example of such an animal model is the transgenic mouse strain is theR6/2 line (Mangiarini et al. Cell 87: 493-506 (1996)). The R6/2 mice aretransgenic Huntington's disease mice, which over-express exon 1 of thehuman HD gene (under the control of the endogenous promoter). The exon 1of the R6/2 human HD gene has an expanded CAG/polyglutamine repeatlengths (150 CAG repeats on average). These mice develop a progressive,ultimately fatal neurological disease with many features of humanHuntington's disease. Abnormal aggregates, constituted in part by the Nterminal part of Huntingtin (encoded by HD exon 1), are observed in R6/2mice, both 45 in the cytoplasm and nuclei of cells (Davies et al. Cell90: 537-548 (1997)). For example, the human Huntingtin protein in thetransgenic animal is encoded by a gene that includes at least 55 CAGrepeats and more preferably about 150 CAG repeats. These transgenicanimals can develop a Huntington's disease-like phenotype.

These transgenic mice are characterized by reduced weight gain, reducedlifespan and motor impairment characterized by abnormal gait, restingtremor, hindlimb clasping and hyperactivity from 8 to 10 weeks afterbirth (for example the R6/2 strain; see Mangiarini et al. Cell 87:493-506 (1996)). The phenotype worsens progressively toward hypokinesia.The brains of these transgenic mice also demonstrate neurochemical andhistological abnormalities, such as changes in neurotransmitterreceptors (glutamate, dopaminergic), decreased concentration ofN-acetylaspartate (a marker of neuronal integrity) and reduced striatumand brain size. Accordingly, evaluating can include assessing parametersrelated to neurotransmitter levels, neurotransmitter receptor levels,brain size and striatum size. In addition, abnormal aggregatescontaining the transgenic part of or full-length human Huntingtinprotein are present in the brain tissue of these animals (e.g., the R6/2transgenic mouse strain). See, e.g., Mangiarini et al. Cell 87: 493-506(1996), Davies et al. Cell 90: 537-548 (1997), Brouillet, FunctionalNeurology 15(4): 239-251 (2000) and Cha et al. Proc. Natl. Acad. Sci.USA 95: 6480-6485 (1998).

To test the effect of the test polypeptide or known polypeptidedescribed in the application in an animal model, differentconcentrations of test polypeptide are administered to the transgenicanimal, for example by injecting the test polypeptide into circulationof the animal. A Huntington's disease-like symptom may be evaluated inthe animal. The progression of the Huntington's disease-like symptoms,e.g., as described above for the mouse model, is then monitored todetermine whether treatment with the test polypeptide results inreduction or delay of symptoms. In another assay, disaggregation of theHuntingtin protein aggregates in these animals is monitored. The animalcan then be sacrificed and brain slices are obtained. The brain slicesare then analyzed for the presence of aggregates containing thetransgenic human Huntingtin protein, a portion thereof, or a fusionprotein comprising human Huntingtin protein, or a portion thereof. Thisanalysis can includes, for example, staining the slices of brain tissuewith anti-Huntingtin antibody and adding a secondary antibody conjugatedwith FITC which recognizes the anti-Huntington's antibody (e.g., theanti-Huntingtin antibody is mouse anti-human antibody and the secondaryantibody is specific for human antibody) and visualizing the proteinaggregates by fluorescent microscopy.

A variety of methods are available to evaluate and/or monitorHuntington's disease. A variety of clinical symptoms and indicia for thedisease are known. Huntington's disease causes a movement disorder,psychiatric difficulties and cognitive changes. The degree, age ofonset, and manifestation of these symptoms can vary. The movementdisorder can include quick, random, dance-like movements called chorea.

Exemplary motor evaluations include: ocular pursuit, saccade initiation,saccade velocity, dysarthria, tongue protrusion, finger tap ability,pronate/supinate, a lo fist-hand-palm sequence, rigidity of arms,bradykinesia, maximal dystonia (trunk, upper and lower extremities),maximal chorea (e.g., trunk, face, upper and lower extremities), gait,tandem walking, and retropulsion. An exemplary treatment can cause achange in the Total Motor Score 4 (TMS-4), a subscale of the UHDRS,e.g., over a one-year period.

Cancer

Methods of the invention which provide administering the Klotho fusionpolypeptide to an individual can be used to treat cancer. Cancerincludes any disease that is caused by or results in inappropriatelyhigh levels of cell division, inappropriately low levels of apoptosis,or both. Examples of cancers include, without limitation, leukemias(e.g., acute leukemia, acute lymphocytic leukemia, acute myelocyticleukemia, acute myeloblastic leukemia, acute promyclocytic leukemia,acute myclomonocytic leukemia, acute monocytic leukemia, acuteerythroleukemia, chronic leukemia, chronic myelocytic leukemia, chroniclymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease,non-Hodgkin's disease), Waldenstrom's macroglobulinemia, heavy chaindisease, and solid tumors such as sarcomas and carcinomas (e.g.,fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, nile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterinecancer, testicular cancer, lung carcinoma, small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodenroglioma, schwannoma,meningioma, melanoma, neuroblastoma, and retinoblastoma).Lymphoproliferative disorders are also considered to be proliferativediseases.

All patents, patent applications, and published references cited hereinare hereby incorporated by reference in their entirety. While thisinvention has been particularly shown and described with references toembodiments thereof, it will be understood by those skilled in the artthat various changes in form and details may be made therein withoutdeparting from the scope of the invention encompassed by the appendedclaims.

5. EXAMPLES Example 1 Expression and Purification of Klotho FusionPolypeptides

Expression of the Klotho Fusion Polypeptide

The polypeptides of the invention were made by transiently transfectingHEK293T cells with an expression vector encoding a Klotho fusionpolypeptide having the extracellular domain of alpha Klotho and theFGF23 (R179Q) variant. Conditioned media containing expressedpolypeptides were generated by transient transfection of the respectiveexpression plasmids for Klotho, FGF23, and the Klotho-FGF23(R179Q)fusion protein. The transfections were performed in 6-well plates usingLipofectamine 2000 (Invitrogen, Cat # 11668-019). Five hours aftertransfection, the transfection mix was replaced with 3 ml DMEM plus 1%FBS. Conditioned media were collected 72 hours after the addition of 3ml DMEM plus 1% FBS. Samples of conditioned medium from varioustransiently transfected HEK293T cells were separated bySDS-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed byWestern blot (FIG. 3A) or stained with Coomassie blue (FIG. 3B).

SDS-polyacrylamide gel electrophoresis was performed on various samples(lane 1, Control; lane 2, FGF23; lane 3, sKlotho; lanes 4-6,sKlotho-FGF23). Coomassie blue staining revealed the expression of ahigh, >180 kDa band (FIG. 3B, indicated by arrow on the right) that wasnot present in lanes 1-3, which contained samples that had not beentransfected with the vector encoding the Klotho fusion polypeptide. Thequality of the Klotho fusion polypeptide secreted into the media wasevaluated by Western blot (FIG. 3A). An anti-FGF23 rat monoclonal IgG2A(R&D Systems, Cat# MAB26291) was used as the primary antibody to detectthe Klotho fusion polypeptides by Western blot. The Western blotconfirmed that the additional bands observed in the Coomassie stainedgels were Klotho fusion polypeptides. The Western blot confirmed thatthe Klotho fusion polypeptides had the expected molecular weight for theKlotho fusion polypeptide. This analysis shows the expression of theKlotho-FGF23(R179Q) fusion protein.

Purification of the Klotho Fusion Polypeptide

The polypeptides of the invention were purified from conditioned mediafrom a culture of HEK293T cells transiently transfected with anexpression vector encoding a Klotho fusion polypeptide having theextracellular domain of alpha Klotho and the FGF23 R179Q variant. Togenerate conditioned medium, an expression vector encodingsKlotho-FGF23-6×His was transfected (500 μg DNA in 18 ml of OptiMEM 1(GIBCO, Cat 11058) mixed with 18 ml of 2 μg/ml polyethlinimine (PEI)into HEK293 cells grown in suspension in expression medium (464 ml ofHEK293T cells at 10⁶ cells/ml in Freestype 293 expression medium (GIBCO,Cat #12338)). After transfection, the culture was allowed to grow (120hours; 37° C. in a 5% CO₂ incubator; shaking at 125 rpm). At the end ofincubation, conditioned medium was harvested by centrifugation (1000 rpmfor five minutes). The conditioned medium was then applied to anickel-agarose column. The sKlotho-FGF23-6×His bound tightly to thecolumn and was eluted with 50 mM imidazole. The resulting purifiedmaterial was then dialyzed in PBS to remove imidazole. A sample of thepurified sKlotho-FGF23-6×His was separated by SDS-PAGE (lane 1, purifiedsKlotho-FGF23-6×His; lane 2, molecular weight marker) and analyzed bystaining with Coomassie blue (FIG. 3C). The stained SDS-PAGE gelconfirmed that the purified sKlotho-FGF23-6×His had the expectedmolecular weight. The inability to detect bands corresponding toproteins other than full-length sKlotho-FGF23-6×His in the lane loadedwith the purified material also showed that the sKlotho-FGF23-6×His waspurified.

Example 2 In Vitro Assay Assessing the Activity of the Klotho FusionPolypeptide

Egr-1-Luciferase

The biological activity of the expressed alpha Klotho fusion polypeptidewas tested in Egr-1-luciferase reporter assays. Binding of the Klothofusion polypeptide to the FGF23 receptor resulted in the downstreamactivation of Egr-1 and the expression of a luciferase reporterregulated by the Egr-1 promoter. The Egr-1-luciferase reporter gene wasconstructed based on that reported by Urakawa et al. (Nature, 2006, Vol444, 770-774). HEK293T cells seeded in 48-well poly-D-lysine plate weretransfected with the Egr-1-luciferase reporter gene together with atransfection normalization reporter gene (Renilla luciferase). Fivehours after transfection of the Egr-1 luciferase reporter gene, thetransfection mix was replaced with 3 ml DMEM plus 1% FBS. Conditionedmedia were collected 72 hours after the addition of 3 ml DMEM plus 1%FBS. Five hours later, the transfection mix was replaced with a sampleto be tested for activity. In initial experiments, 50% conditionedmedium (alone or containing Klotho, FGF23, Klotho and FGF23, and theKlotho-FGF23(R179Q) fusion protein) and 50% DMEM with 1% FBS in thepresence or absence of 20 μg/ml heparin (Sigma, Cat#H8537; dissolved inDMEM as 2 mg ml stock) were tested in the Egr-1-luciferase reporterassays (FIG. 4). Further experiments used defined quantities of thepurified polypeptides (FIGS. 5A and 5B). Cells were lysed 20 hours laterin passive lysis buffer (Promega, Cat #E194A) and luciferase activitieswere determined using Dual-Glo Luciferase Assay System (Promega, Cat#E2940).

In initial experiments, Klotho fusion polypeptide activity wasdemonstrated in unfractionated conditioned medium. Using theEgr-1-luciferase reporter gene (FIG. 4) these experiments quantified thefold changes in the expression of the luciferase reporter. Conditionedmedium containing a combination of FGF23 and the extracellular domain ofKlotho protein activated Egr-1-luciferase, but conditioned mediumcontaining only FGF23 or conditioned medium containing only theextracellular domain of Klotho, did not activate Egr-1-luciferase.Conditioned medium containing the fusion protein sKlotho-FGF23(R179Q)activated the Egr-1-luciferase reporter gene in contrast to conditionedmedia containing either FGF23 or Klotho alone. In these experiments,conditioned medium containing the fusion protein sKlotho-FGF23(R179Q)activated the Egr-1-luciferase reporter gene significantly better thanconditioned medium containing a combination of FGF23 and Klotho. In thepresence of heparin, the inductions by conditioned medium containing thefusion protein sKlotho-FGF23(R179Q) and the conditioned mediumcontaining a combination of FGF23 and Klotho were significantlyenhanced. Table 1 lists the relative expression of various FGF-Klothofusion polypeptides in conditioned medium and the relative activity ofthe unfractionated conditioned medium corresponding to the variousFGF-Klotho fusion polypeptides in Egr-1-luciferase reporter assays.

TABLE 1 Expression and Activities of sKlotho-FGF23 fusion variantsActivity in Egr-1-luc sKlotho-FGF23 fusion constructs Expressionreporter gene 1 sKlotho-FGF23 good yes 2 IgG sp-sKlotho-FGF23 good yes 3sKL-D1-FGF23 good no  4 sKL-D2-FGF23 no n.a. 5 s(KL-D1)2-FGF23 good no 6 sKL-D1/D2-FGF23 no n.a. 7 ssKlotho(ΔN-26)-FGF23 poor no* 8sKLD1-D2(Δ692-965)-FGF23 poor no* 9 sKL-D1-D2(Δ507-798)-FGF23 poor no*10 FGF23-sKlotho poor no* *lack of activity may be the result of lowexpression

Egr-1-luciferase reporter assays were also performed using definedquantities of proteins purified from the conditioned medium, using thepurification procedure as described in Example 1. Consistent withprevious results using unfractionated conditioned medium containing theexpressed polypeptides, treatment with a combination of purified FGF23and sKlotho resulted in luciferase reporter activity, but treatment withpurified FGF23 alone did not (FIG. 5A). The luciferase reporter activityfrom the combination of purified FGF23 and sKlotho was further dependenton the dose of purified sKlotho, and the effect could be enhanced by thepresence of heparin (20 μg ml). An effect of the sKlotho-FGF23-6×Hisfusion polypeptide on luciferase activity could be detected atconcentrations as low as about 1.21 nM (1.2 fold change) and at least upto about 19.3 nM (2.4 fold change) in Egr-1-luciferase reporter assays(FIG. 5B). The activity of the sKlotho-FGF23-6×His fusion polypeptide onluciferase activity was significantly enhanced in the presence ofheparin (20 μg ml). In the presence of heparin, the effect of thesKlotho-FGF23-6×His fusion polypeptide on luciferase activity could bedetected at a concentration as low as about 0.6 nM (2.0 fold change).The result showed that purified sKlotho-FGF23-6×His dose-dependentlyinduced the EGR-1-luc reporter gene, and that treatment withsKlotho-FGF23-6×His.

Example 3 In Vitro Assay Assessing the Effect of the Klotho FusionPolypeptide on Muscle Cells

The biological effect of the expressed Klotho fusion polypeptide wastested on C2C12 myoblasts. Treatment of C2C12 myoblasts with IGF-1,FGF2, or sKlotho-FGF23 resulted in myotube growth and phosphorylation ofsignaling proteins. C2C12 myoblasts were seeded at a density of 40,000cells/well in 6-well poly-D-lysine and fibronectin coated plates ingrowth medium (3 parts DMEM and 1 part F12), 10% FBS, 1% Glut; 1% P/S;1% Linolic acid; 0.1% ITS: [insulin (10 mg ml), transferrin (5.5 mg ml),and selenium (5 ng ml)]. After myoblasts reached confluence (3 days),medium was changed into differentiation medium (DMED with 2% horseserum; 1% Glut; 1% P/S).

For the myotube diameter experiments, three days after confluent mediawas changed into differentiation medium, cells were treated with IGF-1(10 nM), FGF2 (20 ng ml) or sKlotho-FGF23 (20 nM) in the absence orpresence of dexamethasone (100 μM) for 24 hours in differentiationmedium. At the end of treatment, cells were fixed with glutaraldehyde(5% in PBS) and multiple fluorescent images were collected. Myotubediameter was measured using the Pipeline Pilot program to determinehypertrophy or atrophy.

For the signaling protein phosphorylation, experiments, three days afterconfluent media was changed into differentiation medium, cells werestarved for four hours with DMEM without FBS and then treated with IGF-1(10 nM), FGF2 (20 ng/ml) or sKlotho-FGF23 (20 nM) in the absence orpresence of Rapamycin (40 nM) for 30 min. Cells were lysed in RIPAbuffer in the presence of protease and phosphatase inhibitors. Westernblot analysis was carried out and membranes were probed with differentantibodies as indicated in the figure and developed on X-ray films,which were scanned.

The results of this study showed that sKlotho-FGF23 resulted in anincrease in myotube diameter compared to the control and induced C2C12myotube hypertrophy similar to results for IGF-1 and FGF2 (FIG. 5A). Inaddition, treatment with sKlotho-FGF23, IGF-1, and FGF2 could partiallyreverse myotube atrophy induced by dexamethasone, based on measurementsof myotube diameter. No difference was observed between sKlotho-FGF23and FGF2 on myotube morphology (measured by thickness of the myotubes)in the absence or presence of dexamethasone. The trophic effects ofsKlotho-FGF23, IGF-1, and FGF2 were statistically significant.

Consistent with the effects on C2C12 myotubes, sKlotho-FGF23 fusionprotein signaling led to the phosphorylation of p70S6K and ERK, but notAKT or FoxO, in C2C12 myotubes (FIG. 5B). The effect of sKlotho-FGF23 onsignaling was similar to that of FGF2, but was distinct from that ofIGF-1. The extent of ERK phosphorylation by sKlotho-FGF23 was observedto be less than that of IGF-1 or FGF2. The phosphorylation of p70S6K bysKlotho-FGF23 was rapamycin sensitive. In the experiments involvingC2C12 cells, heparin was not required to activate signaling. Theseresults show that a sKlotho-FGF23 fusion polypeptide activated signalingin C2C12 myotubes.

1. A fusion polypeptide comprising: (a) a polypeptide comprising atleast one extracellular subdomain of a Klotho protein; and (b) apolypeptide comprising a fibroblast growth factor.
 2. The fusionpolypeptide of claim 1, wherein the polypeptide of (a) is operativelylinked to the N-terminus of the polypeptide of (b).
 3. The fusionpolypeptide of claim 1, wherein the polypeptide of (b) is operativelylinked to the N-terminus of the polypeptide of (a).
 4. The fusionpolypeptide of claim 1, wherein the polypeptide of (a) and thepolypeptide of (b) are connected by a polypeptide linker.
 5. The fusionpolypeptide of claim 4, wherein the polypeptide linker comprises anamino acid sequence selected from the group consisting of: SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16,SEQ ID NO:17, and SEQ ID NO:18.
 6. The fusion polypeptide of claim 4,wherein the polypeptide linker comprises at least 1 and up to about 30repeats of an amino acid sequence selected from the group consisting of:SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16,SEQ ID NO:17, and SEQ ID NO:18.
 7. The fusion polypeptide of claim 4,wherein the polypeptide of (a) is connected by a peptide bond to theN-terminus of said polypeptide linker, and the polypeptide of (b) isconnected by a peptide bond to the C-terminus of said polypeptidelinker.
 8. The fusion polypeptide of claim 4, wherein the polypeptide of(a) is connected by a peptide bond to the C-terminus of said polypeptidelinker, and the polypeptide of (b) is connected by a peptide bond to theN-terminus of said polypeptide linker.
 9. The fusion polypeptide ofclaim 1, wherein the extracellular subdomain of the Klotho protein is aKL-D1 domain or a KL-D2 domain.
 10. The fusion polypeptide of claim 1,wherein the polypeptide of (a) comprises at least two extracellularsubdomains of the Klotho protein.
 11. The fusion polypeptide of claim10, wherein the two extracellular subdomains of the Klotho protein aretwo KL-D1 domains in tandem repeats.
 12. The fusion polypeptide of claim10, wherein the two extracellular subdomains of the Klotho protein aretwo KL-D2 domains in tandem repeats.
 13. The fusion polypeptide of claim10, wherein the two extracellular subdomains of Klotho protein are aKL-D1 domain and a KL-D2 domain.
 14. The fusion polypeptide of claim 1,wherein the polypeptide of (a) is the extracellular domain of the Klothoprotein.
 15. The fusion polypeptide of claim 1, further comprising asignal peptide.
 16. The fusion polypeptide of claim 15, wherein thesignal peptide is the Klotho signal peptide.
 17. The fusion polypeptideof claim 15, wherein the signal peptide is the IgG signal peptide. 18.The fusion polypeptide of claim 1 that specifically binds to afibroblast growth factor receptor.
 19. The fusion polypeptide of claim1, wherein the Klotho protein is alpha-Klotho.
 20. The fusionpolypeptide of claim 1, wherein the Klotho protein is beta-Klotho. 21.The fusion polypeptide of claim 19, wherein the fibroblast growth factoris fibroblast growth factor-23 or a fibroblast growth factor-23 variant(R179Q).
 22. The fusion polypeptide of claim 20, wherein the fibroblastgrowth factor is fibroblast growth factor-19 or fibroblast growthfactor-21.
 23. The fusion polypeptide of claim 1 comprising an aminoacid sequence which is 95% or more identical to the amino acid sequenceof SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 40, SEQ ID NO:
 41. 24. Thefusion polypeptide of claim 1 having the amino acid sequence of SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:40, or SEQ ID NO:41.
 25. The fusionpolypeptide of claim 1 comprising an amino acid sequence which is 95% ormore identical to the amino acid sequence set forth in SEQ ID NO: 5, SEQID NO: 6, SEQ ID NO: 7, or SEQ ID NO:
 35. 26. A pharmaceuticalcomposition comprising the fusion polypeptide of claim 1 and apharmaceutically acceptable carrier.
 27. A nucleic acid comprising asequence that encodes the fusion polypeptide of claim
 1. 28. A host cellcontaining the nucleic acid of claim
 27. 29. A vector comprising thenucleic acid of claim
 27. 30. A method for treating or preventing anage-related condition in an individual, comprising administering to anindividual in need thereof a therapeutically effective dose of apharmaceutical composition comprising a fusion polypeptide comprising:(a) a polypeptide that comprises at least one extracellular subdomain ofa Klotho protein; and (b) a polypeptide that comprises a fibroblastgrowth factor.
 31. The method of claim 30, wherein the age-relatedcondition is selected from the group consisting of sarcopenia, skinatrophy, muscle wasting, brain atrophy, atherosclerosis,arteriosclerosis, pulmonary emphysema, osteoporosis, osteoarthritis,immunologic incompetence, high blood pressure, dementia, Huntington'sdisease, Alzheimer's disease, cataracts, age-related maculardegeneration, prostate cancer, stroke, diminished life expectancy,memory loss, wrinkles, impaired kidney function, and age-related hearingloss.
 32. The method of claim 30, wherein the Klotho protein is alphaKlotho protein.
 33. The method of claim 31, wherein the age-relatedcondition is muscle wasting, the Klotho protein is alpha Klotho protein,and the fibroblast growth factor is fibroblast growth factor
 23. 34. Amethod for treating or preventing a metabolic disorder in an individual,comprising administering to an individual in need thereof atherapeutically effective dose of a pharmaceutical compositioncomprising a fusion polypeptide, comprising: (a) a polypeptide thatcomprises at least one extracellular subdomain of a Klotho protein; and(b) a polypeptide that comprises a fibroblast growth factor.
 35. Themethod of claim 34, wherein the metabolic disorder is selected from thegroup consisting of Type II Diabetes, Metabolic Syndrome, hyperglycemia,and obesity.
 36. The method of claim 34, wherein the fusion polypeptidecomprises: (a) a polypeptide that comprises at least one extracellularsubdomain of a beta-Klotho protein; and (b) a polypeptide that comprisesa fibroblast growth factor
 21. 37. A method for treating or preventinghyperphosphatemia or calcinosis in an individual, comprisingadministering to an individual in need thereof a therapeuticallyeffective dose of a pharmaceutical composition comprising a fusionpolypeptide, comprising: (a) a polypeptide that comprises at least oneextracellular subdomain of a Klotho protein; and (b) a polypeptide thatcomprises a fibroblast growth factor.
 38. The method of claim 37,wherein the fusion polypeptide comprises: (a) a polypeptide thatcomprises at least one extracellular subdomain of an alpha Klothoprotein; and (b) a polypeptide that comprises a fibroblast growth factor23.
 39. A method for treating or preventing chronic renal disease orchronic renal failure in an individual, comprising administering to anindividual in need thereof a therapeutically effective dose of apharmaceutical composition comprising a fusion polypeptide, comprising:(a) a polypeptide that comprises at least one extracellular subdomain ofa Klotho protein; and (b) a polypeptide that comprises a fibroblastgrowth factor.
 40. The method of claim 39, wherein the Klotho protein isalpha Klotho protein.
 41. A method for treating or preventing cancer inan individual, comprising administering to an individual in need thereofa therapeutically effective dose of a pharmaceutical compositioncomprising a fusion polypeptide, comprising: (a) a polypeptide thatcomprises at least one extracellular subdomain of a Klotho protein; and(b) a polypeptide that comprises a fibroblast growth factor.
 42. Themethod of claim 41, wherein the cancer is breast cancer.
 43. The methodof claim 41, wherein the Klotho protein is an alpha Klotho protein. 44.The fusion polypeptide of claim 1, wherein the Klotho protein is a humanKlotho protein.
 45. The fusion polypeptide of claim 1 for use intreating or preventing muscle atrophy.
 46. A method of treating orpreventing muscle atrophy comprising (consisting essentially of, orconsisting of) administering to an individual in need thereof atherapeutically effective dose of a pharmaceutical compositioncomprising a soluble Klotho protein of SEQ ID NO: 7, SEQ ID NO:44, orSEQ ID NO:45.
 47. A method of treating or preventing muscle atrophycomprising (consisting essentially of, or consisting of) administeringto an individual in need thereof a therapeutically effective dose of apharmaceutical composition comprising (1) a soluble Klotho protein ofSEQ ID NO: 7, SEQ ID NO:44, or SEQ ID NO:45, and (2) a linker.